Methods for identifying and treating resistant tumors

ABSTRACT

This invention provides a method of identifying and reversing multidrug resistance in a multidrug resistant tumor comprising administering a multidrug resistance reversing amount of any of the compounds as defined herein.

BACKGROUND OF THE INVENTION

[0001] Along with surgery and radiotherapy, chemotherapy continues to bean effective therapy for many cancers. In fact, several types of cancerare now considered to be curable by chemotherapy and include Hodgkin'sdisease, large cell lymphoma, acute lymphocytic leukemia, testicularcancer and early stage breast cancer. Other cancers such as ovariancancer, small cell lung and advanced breast cancer, while not yetcurable, are exhibiting positive response to combination chemotherapy.

[0002] One of the most important unsolved problems in cancer treatmentis drug resistance. Drug resistance includes both intrinsic resistanceat the time of treatment using chemotherapy and acquired drugresistance. This problem is a reason for the added importance ofcombination chemotherapy, as the therapy both has to avoid the emergenceof resistant cells and to kill pre-existing cells which are already drugresistant.

[0003] Anthracyclines represent an important class of oncolytic agents.Doxorubicin, an anthracycline, which is also known in the art asAdriamycin™, is a drug of choice in the clinical management of breastcancer. Therapy with anthracyclines such as doxorubicin is complicatedby the appearance of the anthracycline resistant phenotype which limitsor negates the oncolytic activity of doxorubicin.

[0004] Topoisomerase inhibitors represent a further class of oncolyticagents. Epipodophyllotoxins such as Etoposide® and Teniposide® aretopoisomerase inhibitors which are useful in the therapy of neoplasms ofthe testis, small-cell lung and other lung, breast, Hodgkin's disease,non-Hodgkin's lymphomas, acute granulocytic leukemia and Karposi'ssarcoma. The therapeutic utility of the epipodophylotoxins is limited bythe appearance of the epipodophyllotoxin resistant phenotype.

[0005] One form of multi-drug resistance (MDR) is mediated by a 170-180kD energy-dependent efflux pump designated as P-glycoProtein, P-gp. P-gphas been shown to play a major role in the intrinsic and acquiredresistance of a number of human tumors against hydrophobic, naturalproduct drugs. Drugs that act as substrates for and are consequentlydetoxified by P-gp include the vinca alkaloids (vincristine andvinblastine), anthracyclines (Adriamycin), and epipodophyllotoxins(etoposide). While P-gp-associated MDR is a major determinant in tumorcell resistance to chemotherapeutic agents, it is clear that thephenomenon of MDR is multifactorial and involves a number of differentmechanisms. One such alternative pathway for resistance toanthracyclines involves the emergence of a 190 kD protein that is notP-gp. See McGrath, T., Latoud, C., Arnold, S. T., Safa, A. R., Felsted,R. S., and Center, M. S. Biochem. Pharmacol., 38: 3611, (1989). P190,also referred to as MRP, is found on the plasma membrane and alsoappears to be localized in the endoplasmic reticulum See Marquardt, D.and Center, M. S., Cancer Res., 52: 3157, (1992).

[0006] MRP possesses a nucleotide binding domain that is homologous withthe ATP binding site of P-gp. See Marquardt, D., McCrone, S., and CenterM. S., Cancer Res., 50: 1426, (1990). The mechanism(s) utilized by P190to confer resistance to Adriamycin is not well understood but mayinvolve the intracellular redistribution of Adriamycin away from thenucleus. See Marquardt, D. and Center, M. S., supra. Adriamycin is aninhibitor of topoisomerase II (Beck, W. T., Bull. Cancer, 77: 1131,(1990), which is an enzyme involved in DNA replication. Redistributionof Adriamycin away from the nucleus would therefore be an importantcomponent in cellular resistance to this drug. The studies published todate on P190 have utilized cell lines selected in vitro for resistanceto Adriamycin (McGrath, T., Latoud, C., Arnold, S. T., Safa, A. R.,Felsted, R. S., and Center, M. S., supra; Marquardt, D. and Center, M.S., supra; and Marquardt, D., McCrone, S., and Center M. S. Cancer Res.,supra. The association of MRP (P190) with drug resistance was made bysodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ofradioactive extracts prepared from Adriamycin-resistant HL60/Adr humanleukemia cells labeled with 8-azido-alpha[³²P]ATP. See McGrath, T.,Latoud, C., Arnold, S. T., Safa, A. R., Felsted, R. S., and Center, M.S., supra. The drug-resistance phenotype conferred by P190 is notlimited to the anthracyclines. Epipodophyllotoxin resistance is linkedto P190 expression. The IC₅₀s of HL60/S cells treated with Adriamycinand Etoposide were 0.011 μg/ml and 0.39 μg/ml respectively. The IC₅₀sfor HL60/Adr cells (a HL60-derived cell line which is resistant todoxorubicin) treated with Adriamycin and Etoposide were 2.2 μg/mland >10 μg/ml respectively. HL60/S and HL60/Adr cell lines do notexpress P-glycoProtein. HL60/Adr expresses P190. Thus, resistance to theanthracyclines and epipodophyllotoxins results from P190 expression.

[0007] The present invention embraces the elucidation of the role whichMRP plays in multiple drug resistance. MRP functions comprise an energydependent transporter function to transport a range of oncolytics whichinclude compounds which are generally conjugates of lipophilic compoundshaving anionic groups such as cysteinyl, carboxyl, cysteinylglycine,glutatione S, glucuronic acid or sulfate groups.

[0008] Biochemical characterization of the MRP protein allowed thedevelopment of MRP-based assays for compounds which reverse MDR byblocking the transport function of MRP, which in turn led to theidentification of the compounds of the present invention.

SUMMARY OF THE INVENTION

[0009] The invention disclosed and claimed herein provides test kits andassay methodology for measuring MRP inhibition as well as compounds andassociated formulations for use in reversing multiple drug resistanceand therby rendering otherwise refractory neoplasms susceptable tooncolytic therapies. The ability to determine the existance and extentof multiple drug resistance prior to attempts at therapy by evaluating abiopsy sample is a significant advance in the art and allows theelimination of costly and ineffective therapeutic attempts.

[0010] Compounds amenable to use in the invention are generallyamphiphilic anions having a molecular weight of 300 to 950 and amolecular mass of 300 to 950 daltons respectively. Especially preferredmultiple drug resistance compounds for use in accordance with thepresent invention include: MK571, BAY u9773 and compounds of Formula I:

[0011] wherein

[0012] R₁ is

[0013] Y is hydrogen or halo;

[0014] R₂ is hydrogen, —OH, or —OCH₃;

[0015] R₃ is C₁-C₆ alkyl;

[0016] R₄ is hydrogen, —OH, or —OCH₃;

[0017] n is 3, 4, or 5;

[0018] A is

[0019] where

[0020] R₅ is hydrogen, C₁-C₆ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl,benzyl, or phenyl;

[0021] R₆ is hydrogen or halo;

[0022] R₇ is —COOH or 5-tetrazolyl;

[0023] T is a bond, —CH₂—, —O—, —C(═O)—, or —S(O)_(q)—; and

[0024] q is 0, 1, or 2;

[0025] provided when one of R₂ and R₄ is —OH or —OCH₃, the other of R2and R4 must be hydrogen,

[0026] or a pharmaceutically acceptable base addition salt or solvatethereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 shows the ATP-dependent transport of [³H]LTC₄ (left-hand)and S—(2,4-dinitrophenyl)—[³H]glutathione (right-hand side) by membranevesicles of MRP-overexpressing HL60/ADR cells and control cells(cytostatic agent-sensitive parental cells as well as revertants);

[0028]FIG. 2 shows [³H]LTC4-binding proteins and immunoreactive MRP inmembranes of HL60/ADR cells (A), revertant HL60 cells (B) and parental,cytostatic agent-sensitive HL60 cells (C). The top illustrates theevidence of MRP by immunoblot, the molecular weights of standardproteins following electrophoretic separation are shown therebelow, andthe bottom illustrates the introduction of [³H]LTC₄ after photoaffinitylabeling and separation by SDS polyacrylamide electrophoresis. Thesuppression of the [³H]LTC₄ labeling of HL60/ADR membranes by thetransport inhibitor MK571 is shown in the left-hand illustration (A).

[0029]FIG. 3 shows 8-azido[α-³²P] ATP-binding proteins in membranes ofHL60/ADR cells and revertant, cytostatic agent-sensitive cells.

[0030]FIG. 4 shows the inhibition of the ATP-dependent transport of LTC₄by the leukotriene receptor antagonist MK 571. The inhibition is shownin double-reciprocal (Lineweaver and Burk) plot. Membrane vesicles ofMRP-overexpressing cells were incubated in the presence of ATP and LTC₄at 37° C. for 2 minutes. Without the inhibitor (O), the transportactivity is high, as also shown in FIG. 1 (left-hand side). MK 571 (5micromolar) substantially inhibits transport. The inhibition iscompetitive. The inhibition constant (K_(i)) is 0.6 micromolar.

[0031]FIG. 5, Panel A illustrates the ATP-dependent transport of[³H]etoposide glucuronide into membrane vesicles from MRP-overexpressingcells. Panel B provides a comparison of MRP-mediated ATP-dependenttransport of [³H]etoposide glucuronide and [³H]etoposide into membranevesicles from MRP-overexpressing cells. Panel C provides a comparison ofMRP-mediated ATP-dependent transport of [³H]etoposide glucuronide intomembrane vesicles from MRP-overexpressing cells and controls cells andits inhibition by 5 μM MK 571. The structures of etoposide and etoposideglucuronide are provided in Panel D.

[0032]FIG. 6 illustrates the ATP-dependent transport of LTC4 intomembrane vesicles from MRP-overexpressing cells and its competitiveinhibition by the leukotriene receptor antagonist BAY u9773. Theinhibition constant (K_(i)) is 0.7 micromolar

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present inventors have determined that a membrane proteinwhich mediates the ATP-dependent transport of certain conjugates such asglucuronides and glutathione S-conjugates is associated with thecytostatic-agent resistance of certain tumors. The terms cytostaticagent, cytotoxic agent and oncolytic are used interchangeably todescribe the present invention and these terms encompass the compoundsused in cancer therapy regimens.

[0034] Elucidation of the substrate specificity of MRP allowed theselection and characterization of numerous compounds having multipledrug resistance reversal activity. Amphiphilic anions which arelipophilic compounds having a negative charge and a molecular weight of300 to 950 (daltons), represent substrates for MRP and inhibit itsability to transport cytostatic agents out of the tumor cell again. Ofparticular interest are compounds which are usually conjugates oflipophilic compounds having anionic groups such as cysteinyl, carboxyl,cysteinylglycine, glutathione S, glucuronic acid or sulfate groups. Theglutathione S-conjugate leukotriene C₄ as well as leukotrienes D₄ and E₄were identified as substrates for the MRP protein, occurring naturallyin the body. For this reason, substances structurally related to theleukotrienes C₄, D₄ and E₄ (LTC₄, LTD₄, LTE₄) are effective inhibitorsfor MRP. For example, cysteinyl leukotriene receptor antagonists are inconsideration as substances structurally related to the leukotrienes.The receptor antagonists which react with C_(4,) D₄ and E₄ leukotrienereceptors are also understood to include compounds structurally relatedto the receptor antagonists. The use of these inhibitors leads to aninhibition of MRP and thus to a drastic reduction of thecytostatic-agent resistance in tumors, e.g. in the case of bronchialcarcinomas and leukemias. The compounds relevant to the presentinvention are grouped for description based on the extensive mechanisticdata and assay kit development data which was generated using MK 571 andBAY u9773 versus the extensive synthetic chemistry effort directed tothe phenoxy compounds of Formula I. Examples 1-18 describe the chemistryand cell-based assay system activity data relevant to the phenoxycompounds of Formula I.

[0035] A subset of leukotriene receptor antagonists of particularimportance are those described in the scientific and patent literaturein association with the bronchodilatation of asthma, e.g. MK 571 and BAYu9773. These compounds and their use in elucidating the mechanism of MRPand assay and kit development are further described below and inExamples 19-21 Results of transporter assays are summarized in FIGS.1-6. The studies summarized in FIGS. 1-6 indicate that the preferred ofthe inhibitor at the site of action is 0.1 to 10 micromolar. The presentinventors have further determined that a concentration of a MDR reversalagent of about 0.5 to to about 50 mg/kg body weight of the patientshould be given to achieve the desired concentration at the site ofaction.

[0036] The methodology described in the Examples for evaluating variouscompounds for their ability to compete with radiolabeled leukotrienesfor transport enables the preparation of various kits for measuringtransport function or inhibition. Those of ordinary skill in the artwill appreciate that various other “detector” groups such asfluorescent-labeled leukotrienes can be used in lieu of radioisotopesfor providing a means to measure leukotriene transport or the inhibitionthereof.

[0037] The following definitions refer to the various terms usedthroughout this disclosure to further describe the compounds of FormulaI. The term “C₁-C₆ alkyl” refers to the straight and branched aliphaticradicals of 1 to 6 carbon atoms such as methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, 2,2-dimethylpropyl,hexyl, and the like. Included within this definition are the terms“C₁-C₃ alkyl”, “C₁-C₄ alkyl” and “C₁-C₅ alkyl”. The term “C₂-C₅ alkenyl”refers to straight and branched aliphatic radicals of 2 to 5 carbonatoms containing one double bond, such as —CH═CH₂, —CH₂CH═CH₂,—CH₂CH₂CH═CH₂, —CH₂C(CH₃)═CH₂, —CH₂CH═C(CH₃)₂, and the like. The term“C₂-C₅ alkynyl” refers to straight and branched aliphatic residues of 2to 5 carbon atoms containing one triple bond, such as —C≡CH, —CH₂—C≡CH,—CH₂CH₂C≡CH, —CH₂CH(CH₃)C≡CH, —CH₂C≡CCH₃, and the like. The term “halo”refers to fluoro, chloro, bromo, and iodo.

[0038] Preferred compounds of Formula I which are employed in thisinvention are those wherein R₁ is 4-halo (especially fluoro)phenyl oracetyl. In particular, compounds wherein

[0039] R₁-R₄-substituted phenyl is

[0040]

[0041] are preferred, especially when R₃ is propyl or ethyl. It is alsopreferred that R₇ is —COOH and R₅ is either propyl or benzyl. Compoundswherein

[0042] A is

[0043] are especially preferred.

[0044] This invention includes the use of pharmaceutically acceptablebase addition salts of the compounds of Formula I. Such salts includethose derived from inorganic bases, such as ammonium and alkali andalkaline earth metal hydroxides, carbonates, bicarbonates, and the like,as well as salts derived from basic organic amines, such as aliphaticand aromatic amines, aliphatic diamines, hydroxy alkylamines, and thelike. Such bases useful in preparing the salts of this invention thusinclude ammonium hydroxide, potassium carbonate, sodium bicarbonate,calcium hydroxide, methyl amine, diethyl amine, ethylene diamine,cyclohexylamine, ethanolamine, and the like. The potassium and sodiumsalt forms are particularly preferred. In addition, this inventionincludes any solvate forms of the compounds of Formula I or saltsthereof, such as ethanol solvates, hydrates, and the like.

[0045] It is recognized that in compounds having a branched alkylfunctionality, and in those compounds bearing double or triple bonds,various stereoisomeric products may exist. This invention is not limitedto any particular stereoisomer but includes all possible individualisomers and mixtures thereof. The term “5-tetrazolyl” refers to bothtautomers, i.e., (1H)-5-tetrazolyl and (2H)-5-tetrazolyl.

[0046] An important aspect of the current invention concerns thediscovery that a select group of phenoxy compounds, those of Formula I,and the racemate and isomers of MK 571 and Bay u9771 are useful fortreating resistant neoplasms. The synthesis of compounds of Formula 1are taught in Examples 1-18. BAY u977 is commercially avialable fromCascade Biochem LTD, University Park, Whiteknights, Reading, BerkshireRG6 6BX, United Kingdom. MK571 and structurally related compounds of usein the present invention are taught in U.S. Pat. No. 5,350,761; U.S.Pat. No. 5,260,316; EP 526402-A1; U.S. Pat. No. 5,310,884; U.S. Pat. No.5,155,130; EP 412,939-A; U.S. Pat. No. 5,120,758; U.S. Pat. No.5,118,858; U.S. Pat. No. 5,428,171; U.S. Pat. No. 4,851,409; and EP233763, the entire contents of which are hereby incorporated byreference. The methods of treatment provided by this invention arepracticed by administering to a human or other mammal in need amultidrug resistance reversing amount of a compound of Formula I or apharmaceutically acceptable salt or solvate thereof, that is effectiveto make the neoplasms less resistant to chemotherapy. In making theneoplasm less resistant, the compounds of the invention may be used onneoplasms having intrinsic and/or acquired resistance. Such neoplasmsinclude those which have a pathway for resistance which includes theprotein P190 (MRP). Resistance to drugs such as epipodophyllotoxins andanthracyclines are linked to P190. The treatment of the resistant andsusceptible neoplasm will result in a reversal or inhibition ofresistance, or in other words, will cause the neoplasm to be moresensitive to the appropriate chemotherapy such as treatment withvinblastine, vincristine, vindesine, navelbine, daunorubicin,doxorubicin, mitroxantrone, etoposide, teniposide, mitomycin C,actinomycin D, taxol, topotecan, mithramycin, colchicine, puromycin,podophyllotoxin, ethidium bromide, emetine, gramicidin D, andvalinomycin.

[0047] The compounds of the invention may be used for many resistantneoplasms, including colon cancer, mesothelioma, melanoma, prostatecancer, ovarian cancer, non-small cell lung cancer, small-cell lungcancer, bladder cancer, endometrial cancer, leukemia, testicular cancer,breast cancer, and large cell lymphoma. More particular types of cancerare Hodgkin's disease, Karposi's sarcoma, and acute granulocyticleukemia.

[0048] Generally, the compound is formulated with common excipients,diluents or carriers, and compressed into tablets, or formulated aselixirs or solutions for convenient oral administration, or administeredby the intramuscular or intravenous routes. The compounds can beadministered transdermally, and may be formulated as sustained releasedosage forms and the like. Formulations containing more than one of thecompounds of the present invention are also contemplated for use in themethods of the present invention.

[0049] The compounds of Formula I which can be beneficially used in themethods of the current invention can be made according to establishedprocedures, such as those detailed in U.S. Pat. Nos. 5,324,743 and4,889,871 and EPO Patent Applcation Publication 544,488, all of whichare incorporated by reference herein.

[0050] The phenoxy compounds of Formula I employed in this invention areeither disclosed in the two aforementioned references or can be preparedaccording to the same methods as disclosed therein. Other references forpreparing other related compounds of this type, generally known asleukotriene antagonists, also provide the skilled organic chemist withmethods for preparing such compounds.

[0051] The following preparations and examples further illustrate thepreparation of the compounds employed in this invention. The examplesare illustrative only and are not intended to limit the scope of theinvention. Melting points were determined on a Thomas-Hoover apparatusand are uncorrected. NMR spectra were determined on a GE QE-300spectrometer. All chemical shifts are reported in parts per million (∂)relative to tetramethylsilane. The following abbreviations are used todenote signal patterns: s=singlet, d=doublet, t=triplet, q=quartet,b=broad, m=multiplet. Infrared spectra were determined on a Nicolet DX10FT-IR spectrometer. Mass spectral data were determined on a CEC-21-110spectrometer using electron impact (EI) conditions, a MAT-731spectrometer using free desorption (FD) conditions, or a VG ZAB-3Fspectrometer using fast atom bombardment (FAB) conditions. Silica gelchromatography was performed using ethyl acetate/hexane gradients unlessotherwise indicated. Reverse-phase chromatography was performed on MCICHP20P gel using an acetonitrile/water or methanol/water gradient unlessotherwise indicated. Tetrahydrofuran (THF) was distilled fromsodium/benzophenone ketyl immediately prior to use. All reactions wereconducted under argon atmosphere with stirring unless otherwise noted.Where structures were confirmed by infra-red, proton nuclear magneticresonance, or mass spectral analysis, the compound is so designated by“IR”, “NMR”, or “MS”, respectively.

Example 18-Propyl-7-[3-[4-(4-fluorophenyl)-2-ethyl-5-hydroxyphenoxy]propoxy]-3,4-dihydro-2H-1-benzopyran-2-carboxylicacid

[0052]

[0053] A. Preparation of ethyl 8-propyl-7-[3-[2-ethyl-4-(4-fluorophenyl)-5-benzyloxyphenoxy]propoxy]-3,4-dihydro-2H-1-benzopyran-2-carboxylate.

[0054] Tetrakis(triphenylphosphine)palladium(0) (0.659 g, 0.6 mmol) andaqueous sodium carbonate solution (20 mL of a 2M solution) were added toa 30 mL benzene solution of ethyl7-[3-[(2-benzyloxy-1-bromo-5-ethyl-4-yl)oxy]propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylate(2.163 g, 3.5 mmol) under an argon atmosphere. The reaction was refluxedfor 17 hours, then cooled to room temperature and extracted with ethylacetate. The organic extract was dried over magnesium sulfate, filteredand the solvent removed under vacuum. The crude product was purified byWaters Prep 500 silica gel chromatography eluting with a gradient of 5%to 20% ethyl acetate/hexane over 50 minutes. The desired title biphenylwas obtained as a clear oil (1.722 g, 78%). NMR (CDCl₃) 7.51 (m, 2),7.32 (m, 5), 7.09 (m, 3), 6.83 (d, 1, J=8.32 Hz), 6.62 (s, 1), 6.49 (d,1, J=8.50 Hz), 5.02 (s, 2), 4.75 (dd, 1, J=4.10, 6.50 Hz), 4.22 (m, 6),2.69 (m, 6), 2.25 (m, 4), 1.59 (m, 2), 1.30 (t, 3, J=7.10 Hz), 1.21 (t,3, J=7.42 Hz), 0.96 (t, 3, J=7.33 Hz); IR (CHCl₃) 3019, 2968, 1745,1611, 1495 cm⁻¹; Mass Spec. (FAB) (m/z) 627 (M++1), 626 (M+), 536.

[0055] Analysis for C₃₉H₄₃O₆: Calc: C, 74.74; H, 6.91; F, 3.03; Found:C, 74.98; H, 7.05; F, 3.39.

[0056] B. Preparation of ethyl 8-propyl-7-[3-[4-(4-fluorophenyl)-2-ethyl-5-hydroxyphenoxy]propoxy]-3,4-dihydro-2H-1-benzopyran-2-carboxylate.

[0057] Hydrogen gas was bubbled for 10 minutes through a solution ofethyl 8-propyl-7-[3-[2-ethyl-4-(4-fluorophenyl)-5-benzyloxy-phenoxy]propoxy]-3,4-dihydro-2H-1-benzopyran-2-carboxylate(1.610 g, 2.57 mmol) in 30 mL of ethyl acetate containing 1.0 g of 10%Pd/C catalyst. The reaction was stirred at room temperature under anatmosphere of hydrogen for 2 hours. The reaction mixture was filteredthrough a Celite® pad in a sintered glass funnel and the catalyst waswashed with ethyl acetate. The solvent was removed from the filtrateproviding 1.242 g of a clear oil. The oil was purified by flashchromatography on Merck silica gel eluting with 20% ethylacetate/hexane. The desired title phenol was obtained in 74% yield(1.020 g) as a white solid. TLC: Rf=0.35 (30% ethyl acetate/hexane) NMR(CDCl₃) 7.43 (m, 2), 7.16 (dd, 2, J=5.97, 5.97 Hz), 6.98 (s, 1), 6.82(d, 1, J=8.44 Hz), 6.53 (s, 1), 6.46 (d, 1, J=9.43 Hz), 5.07 (s, 1),4.76 (m, 1), 4.21 (m, 6), 2.67 (m, 6), 2.26 (m, 4), 1.58 (m, 2), 1.29(t, 3, J=6.96 Hz), 1.91 (t, 3, J=7.35 Hz), 0.96 (t, 3, J=7.27 Hz); IR(KBr) 3434, 2962, 2869, 1738, 1614, 1588, 1502 cm⁻¹; Mass Spec (FAB)(m/z) 537 (M++1), 536 (M+).

[0058] Analysis for C₃₂H₃₇O₆: Calc: C, 71.62; H, 6.95; Found: C, 71.63;H, 7.06.

[0059] C. Preparation of 8-propyl-7-[3-[4-(4-fluorophenyl)-2-ethyl-5-hydroxyphenoxy]propoxy]-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid.

[0060] A dioxane (12 mL) solution of ethyl8-propyl-7-[3-[4-(4-fluorophenyl)-2-ethyl-5-hydroxyphenoxy]propoxy]-3,4-dihydro-2H-1-benzopyran-2-carboxylate (0.968 g, 1.8 mmol) was treated withsodium hydroxide (2.71 mL of a 2N solution) and stirred at roomtemperature. After 2.5 hours at room temperature, the dioxane wasremoved from the reaction mixture and the remaining material was dilutedwith water and acidified to pH 1 with 5N hydrochloric acid. Theresulting white milky suspension was then stirred with ethyl acetate andsubsequently extracted with ethyl acetate. The organic extract was driedover magnesium sulfate, filtered and the solvent removed to give a whitesolid (1.098 g). The solid was recrystallized from ethyl acetate/hexaneto give the title acid as white needle-like crystals (0.568 g, 62%).TLC: Rf 0.31 (10% methanol/methylene chloride) NMR (CDCl₃) ∂ 7.42 (m,2), 7.15 (dd, 2, J=8.68), 6.98 (s, 1), 6.85 (d, 1, J=8.30 Hz), 6.53 (s,1), 6.52 (d, 1, J=6.98 Hz), 4.77 (dd, 1, J=3.63, 7.43 Hz), 4.18 (m, 4),2.70 (m, 6), 2.27 (m, 4), 1.56 (m, 2), 1.19 (t, 3, J=7.42 Hz), 0.95 (t,3, J=7.30 Hz); IR (KBr) 3421, 2959, 2871, 1706, 1615, 1500 cm⁻¹; MassSpec (FAB) (m/z) 509 (M++1), 508 (M+).

[0061] Analysis for C₃₀H₃₃O₆: Calc: C, 70.78; H, 6.54; Found: C, 70.05;H, 6.82.

Example 22-[2-Propyl-3-[3-(2-ethyl-5-hydroxy-4-phenylphenoxy)-propoxy]phenoxy]benzoicacid sodium salt hemihydrate

[0062]

[0063] A. Preparation of 2-(3-hydroxy-2-propylphenoxy)-benzoic acidmethyl ester.

[0064] A mixture of 1,3-dihydroxy-2-propylbenzene (75.0 g, 0.490 mol),methyl 2-iodobenzoate (129 g, 0.490 mol), copper bronze (47.0 g, 0.740mol) and potassium carbonate (81.7 g, 0.592 mol) in dry pyridine (1 L)was thoroughly de-gassed with nitrogen, then refluxed for 6 hours. Themixture was cooled to room temperature, filtered, and concentrated invacuo to reveal a dark sludge. This material was dissolved in ethylacetate and passed down a short (˜500 cm³) Florisil® column. Theresulting solution was washed twice with a saturated copper sulfatesolution and concentrated in vacuo. The residue was dissolved inmethylene chloride, washed once with a 0.5 N sodium hydroxide solution,and washed once with a dilute sodium hydroxide solution. The organiclayer was dried over sodium sulfate, filtered, and concentrated in vacuoto provide a clear brown oil. Silica gel chromatography (ethylacetate/hexane) provided 45.4 g (32%) of the desired title intermediateas a white solid: mp 80° C.; NMR (CDCl₃) 7.92 (dd, J=7.8, 1.6 Hz, 1H),7.42 (t, J=8.4 Hz, 1H), 7.13 (t, J=7.2 Hz, 1H), 6.97 (t, J=8.1 Hz, 1H),6.86 (d, J=8.1 Hz, 1H), 6.62 (d, J=8.0 Hz, 1H), 6.51 (d, J=8.0 Hz, 1H),5.65 (bs, 1H, —OH), 3.88 (s, 3H), 2.66 (t, J=7.6 Hz, 2H), 1.62 (hextet,J=7.6 Hz, 2H), 0.96 (t, J=7.4 Hz, 3H); MS-FD m/e 286 (p); IR (CHCl₃,cm⁻¹) 3350 (b), 2950, 1718, 1602, 1480, 1306, 1255, 1086, 981.

[0065] Analysis for C₁₇H₁₈O₄: Calc: C, 71.31; H, 6.34; Found: C, 71.53;H, 6.37.

[0066] B. Preparation of 2-[2-propyl-3-[3-(2-ethyl-5-hydroxy-4-phenylphenoxy)propoxy]phenoxy]benzoic acid sodium salt hemihydrate.

[0067] 2-(3-Hydroxy-2-propylphenoxy)benzoic acid methyl ester (450 mg,1.57 mmol) was alkylated with 2-benzyloxy-1-phenyl-5-ethyl-4-(3-chloro-1-propyloxy)benzene, de-benzylated, and hydrolyzed.Salt formation and purification provided 200 mg (21%) of the desiredtitle product as a fluffy white solid: NMR (DMSO-d₆) 7.48 (d, J=7.5 Hz,2H), 7.42 (d, J=7.2 Hz, 1H), 7.31 (t, J=7.4 Hz, 2H), 7.18 (t, J=7.5 Hz,1H), 7.16 (t, J=7.1 Hz, 1H), 6.98 (m, 3H), 6.64 (t, J=7.2 Hz, 2H), 6.60(s, 1H), 6.24 (d, J=7.9 Hz, 1H), 4.15 (m, 2H), 4.02 (m, 2H), 2.61 (m,2H), 2.49 (m, 2H), 2.16 (t, J=5.5 Hz, 2H), 1.46 (hextet, J=6.6 Hz, 2H),1.07 (t, J=7.4 Hz, 3H), 0.82 (t, J=7.4 Hz, 3H); MS-FAB m/e 549 (100, p+1), 526 (32), 295 (28), 252 (34), 227 (20), 213 (21); IR (CHCl₃, cm⁻¹)3450 (b), 2974, 1602, 1586, 1461, 1393, 1240, 1113, 1048.

[0068] Analysis for C₃₃H₃₂O₆Na·0.5 H₂O: Calc: C, 71.22; H, 5.94; Found:C, 71.42; H, 6.16.

Example 35-Ethyl-4-[3-[2-propyl-3-[2-(2H-tetrazol-5-yl)phenoxy]phenoxy]propoxy][1,1′-biphenyl]-2-oldisodium salt sesquihydrate

[0069]

[0070] A. Preparation of 3-(2-cyanophenoxy) -2-propylphenol.

[0071] A mixture of 3-hydroxy-2-propylphenol (7.50 g, 49.3 mmol),2-bromobenzonitrile (8.97 g, 49.3 mmol), copper bronze (3.76 g, 59.2mmol), and potassium carbonate (6.80 g, 49.3 mmol) in pyridine (250 mL)was refluxed for 72 hours. The mixture was cooled to room temperature,filtered, and concentrated in vacuo. The residue was dissolved in ethylacetate and washed once with water and three times with a saturatedcopper sulfate solution. The organic layer was dried over sodiumsulfate, filtered, and concentrated in vacuo to provide a dark oil.Silica gel chromatography provided a white solid. Sublimation of thismaterial (bulb-to-bulb distillation apparatus, 200° C.) to remove excess3-hydroxy-2-propylphenol provided 1.79 g (14%) of the desired titleintermediate as an off-white crystalline material: mp 103-107° C.; NMR(CDCl₃) 7.68 (d, J=8 Hz, 1H), 7.47 (t, J=7 Hz, 1H), 7.12 (t, J=8 Hz,1H), 7.10 (t, J=8 Hz, 1H), 6.80 (d, J=9 Hz, 1H), 6.71 (d, J=9 Hz, 1H),6.58 (d, J=9 Hz, 1H), 4.95 (s, 1H, —OH), 2.62 (t, J=7 Hz, 2H), 1.60(hextet, J=6 Hz, 2H), 0.96 (t, J=7 Hz, 3H); MS-FD m/e 253 (p); IR(CHCl₃, cm⁻¹) 3300 (b), 2967, 2234, 1600, 1485, 1483, 1450, 1247, 1097,980.

[0072] Analysis for C₁₆H₁₅NO₂: Calc: C, 75.87; H, 5.97; N, 5.53; Found:C, 75.09; H, 5.88; N, 5.58.

[0073] B. Preparation of 5-ethyl-4-[3-[2-propyl-3-[2-(2H-tetrazol-5-yl)phenoxy]phenoxy]propoxy][1,1′-biphenyl]-2-ol disodium saltsesquihydrate.

[0074] 3-(2-Cyanophenoxy)-2-propylphenol (1.66 g, 6.56 mmol) wasalkylated with 2-benzyloxy-1-phenyl-5-ethyl-4-(3-chloro-1-propyloxy)benzene. The crude product was dissolved in hexane/ethyl acetate andpassed through a short silica gel column. The solution was concentratedin vacuo and the residue dissolved in 2-methoxyethanol (50 mL). To thissolution was added lithium azide (1.38 g, 24.2 mmol) andtriethylammonium bromide (1.30 g, 7.14 mmol). The resulting mixture wasrefluxed for 48 hours, cooled to room temperature, and passed down ashort silica gel column. The column was washed with excess ethyl acetateand the combined washings were concentrated in vacuo. The resultingmaterial was de-benzylated and the crude tetrazole was converted to thesodium salt and purified to provide 320 mg (8%) of the desired titleproduct as a fluffy white solid: NMR (DMSO-d₆) 7.81 (dd, J=7.7, 1.5 Hz,1H), 7.49 (d, J=7.5 Hz, 2H), 7.33 (t, J=7.5 Hz, 2H), 7.21 (m, 2H), 7.11(t, J=7.3 Hz, 1H), 6.99 (m, 2H), 6.76 (d, J=8.1 Hz, 1H), 6.68 (d, J=8.2Hz, 1H), 6.56 (s, 1H), 6.22 (d, J=8.2 Hz, 1H), 4.16 (t, J=5.8 Hz, 2H),4.10 (t, J=5.9 Hz, 2H), 2.61 (t, J=6.5 H, 2H), 2.48 (m, 2H), 2.22 (m,2H), 1.45 (hextet, J=7.4 Hz, 2H), 1.08 (t, J=7.4 Hz, 3H), 0.79 (t, J=7.3Hz, 3H); MS-FAB m/e 595 (35, p +1), 574 (39), 573 (100), 551(99); IR(KBr, cm⁻¹) 3418 (b), 2962, 1577, 1458, 1243, 1229, 1147, 1117.

[0075] Analysis for C₃₃H₃₂N₄O₄Na₂·1.5 H₂O: Calc: C, 63.76; H, 5.68; N,9.01; Found: C, 63.63; H, 5.59; N, 8.80.

Example 4 2-Fluoro-6-[2-propyl-3-[3-(2-ethyl-5-hydroxy-4-phenylphenoxy)propoxy]phenoxy]benzoic acid disodium salt

[0076]

[0077] A. Preparation of 2-fluoro-6-(3-hydroxy-2-propyl-phenoxy) benzoicacid methyl ester.

[0078] 2-Fluoro-6-iodobenzoic acid methyl ester (13.1 g, 46.8 mmol) wassubmitted to the Ullmann conditions. This procedure provided 3.10 g(22%) of the desired title intermediate as an oil: NMR (CDCl₃) 7.26 (m,1H), 7.03 (t, J=8.1 Hz, 1H), 6.83 (t, J=8.6 Hz, 1H), 6.65 (d, J=8.0 Hz,1H), 6.56 (d, J=7.8 Hz, 1H), 6.53 (d, J=7.6 Hz, 1H), 5.30 (bs, 1H, —OH),3.93 (s, 3H), 2.59 (t, J=7.3 Hz, 2H), 1.56 (hextet, J=7.6 Hz, 2H), 0.94(t, J=7.4 Hz, 3H).

[0079] B. Preparation of 2-fluoro-6-[2-propyl-3-[3-(2-ethyl-5-hydroxy-4-phenylphenoxy)propoxy]phenoxy]benzoic acid disodium salt.

[0080] 2-Fluoro-6-(3-hydroxy-2-propylphenoxy)benzoic acid methyl ester(0.660 g, 2.17 mmol) was alkylated with 2-benzyloxy-1-phenyl-5-ethyl-4-(3-chloro-1-propyloxy)benzene to provide crudeproduct as an oil. The oil was de-benzylated, hydrolysed, salified, andpurified to provide 468 mg (37%) of the desired title product as afluffy white solid: NMR (DMSO-d₆) 7.49 (d, J=8.8 Hz, 2H), 7.32 (t, J=7.5Hz, 2H), 7.18 (t, J=7.4 Hz, 1H), 6.85-7.10 (m, 3H), 6.74 (t, J=8.1 Hz,2H), 6.62 (s, 1H), 6.42 (d, J=8.1 Hz, 1H), 6.33 (d, J=8.2 Hz, 1H), 4.13(t, J=6.0 Hz, 2H), 4.04 (t, J=5.8 Hz, 2H), 2.40-2.63 (m, 4H), 2.15 (m,2H), 1.41 (hextet, J=7.3 Hz, 2H), 1.07 (t, J=7.4 Hz, 3H), 0.79 (t, J=7.2Hz, 3H); MS-FAB m/e 589 (16, p), 568 (36), 567 (100), 546 (30), 527(15); IR (CHCl₃, cm⁻¹) 2975, 1601, 1456, 1395, 1115, 1047.

[0081] Analysis for C₃₃H₃₁O₆FNa₂: Calc: C, 67.34; H, 5.31; F, 3.23;Found: C, 67.43; H, 5.59; F, 2.99.

Example 52-[2-Propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]phenoxy]benzoicacid sodium salt

[0082]

[0083] A. Preparation of 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]propoxy]phenoxy]-benzoic acid methyl ester.

[0084] A mixture of2-benzyloxy-l-(4-fluorophenyl)-5-ethyl-4-(3-chloro-l-propyloxy)benzene(20.0 g, 50.2 mmol) and sodium iodide (75.3 g, 502 mmol) in 2-butanone(200 mL) was refluxed for 6 hours. The mixture was diluted with etherand washed once with water. The organic layer was dried over sodiumsulfate, filtered, and concentrated in vacuo to provide a colorless oil.This material was dissolved in dimethylformamide (100 mL) and treatedwith 2-(3-hydroxy-2-propylphenoxy)benzoic acid methyl ester (14.4 g,50.2 mmol) and potassium carbonate (20.8 g, 151 mmol) at roomtemperature for 24 hours. This mixture was diluted with water and twiceextracted with ether. The aqueous layer was separated and back-extractedonce with ethyl acetate. The combined organic layers were dried oversodium sulfate, filtered, and concentrated in vacuo to provide a yellowoil. Silica gel chromatography provided 25.4 g (78%) of the desiredtitle intermediate as a pale golden oil: NMR (CDCl₃) 7.91 (∂, J=7.8 Hz,1H), 7.54 (d, J=8.6 Hz, 1H), 7.52 (d, J=8.5 Hz, 1H), 7.25-7.43 (m, 6H),7.03-7.38 (m, 5H), 6.84 (d, J=8.3 Hz, 1H), 6.71 (d, J=8.1 Hz, 1H), 6.63(s, 1H), 6.47 (d, J=8.1 Hz, 1H), 5.03 (s, 2H), 4.24 (t, J=5.7 Hz, 2H),4.21 (t, J=5.8 Hz, 2H), 3.86 (s, 3H), 2.69 (t, J=7.8 Hz, 2H), 2.64 (t,J=7.7 Hz, 2H), 2.34 (quintet, J=6.0 Hz, 2H), 1.60 (hextet, J=5.0 Hz,2H), 1.22 (t, J=7.5 Hz, 3H), 0.94 (t, J=7.5 Hz, 3H); MS-FD m/e 648 (p);IR (CHCl₃, cm⁻¹) 2960, 1740, 1604, 1497, 1461, 1112.

[0085] Analysis for C₄₁H₄₁O₆F: Calc: C, 75.91; H, 6.37; Found: C, 76.15;H, 6.45.

[0086] B. Preparation of 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]phenoxy]benzoic acid methyl ester.

[0087] 2-[2-Propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]propoxy]phenoxy]benzoic acid methyl ester (33.0 g, 50.9 mmol)was de-benzylated to provide 27.3 g (96%) of the title intermediate asan amber oil: NMR (CDCl₃) 7.90 (dd, J=7.8, 1.7 Hz, 1H), 7.42 (m, 3H),7.05-7.23 (m, 4H), 6.99 (s, 1H), 6.84 (d, J=8.1 Hz, 1H), 6.70 (d, J=8.1Hz, 1H), 6.55 (s, 1H), 6.46 (d, J=8.1 Hz, 1H) , 5.05 (s, 1H, —OH), 4.23(m, 4H) , 3.86 (s, 3H) , 2.68 (t, J=7.4 Hz, 2H), 2.62 (q, J=7.5 Hz, 2H),2.36 (quintet, J=6.0 Hz, 2H), 1.60 (hextet, J=7.7 Hz, 2H), 1.20 (t,J=7.6 Hz, 3H), 0.94 (t, J=7.4 Hz, 3H); MS-FD m/e 558 (p); IR (CHCl₃,cm⁻¹) 2965, 1727, 1603, 1496, 1458, 1306, 1112.

[0088] Analysis for C₃₄H₃₅O₆F: Calc: C, 73.10; H, 6.31; Found: C, 73.17;H, 6.42.

[0089] C. Preparation of 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]phenoxy]benzoic acid sodium salt.

[0090]2-[2-Propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]phenoxy]benzoicacid methyl ester (21.5 g, 38.5 mmol) was hydrolyzed. The resulting acidwas converted to the sodium salt and purified to provide 16.7 g (77%) ofthe desired title product as a white amorphous solid: NMR (DMSO-d₆)10.50 (bs, 1H, —OH), 7.51 (m, 3H), 7.20 (t, J=7.4 Hz, 1H), 7.13 (m, 2H),7.00 (m, 2H), 6.95 (s, 1H), 6.67 (dd, J=8.2, 3.3 Hz, 2H), 6.62 (s, 1H),6.26 (d, J=8.2 Hz, 1H), 4.14 (t, J=5.8 Hz, 2H), 4.02 (t, J=5.7 Hz, 2H),2.60 (t, J=6.8 Hz, 2H), 2.47 (q, J=7.3 Hz, 2H), 2.16 (t, J=5.9 Hz, 2H),1.45 (hextet, J=7.5 Hz, 2H), 1.07 (t, J=7.5 Hz, 3H), 0.81 (t, J=7.4 Hz,3H); MS-FAB m/e 568 (38, p +1), 567 (100, p), 544 (86), 527 (77), 295(65), 253 (45); IR (KBr, cm⁻¹) 3407 (b), 2962, 1603, 1502, 1446, 1395,1239, 1112.

[0091] Analysis for C₃₃H₃₂O₆FNa: Calc: C, 69.95; H, 5.69; F, 3.35;Found: C, 69.97; H, 5.99; F, 3.52.

Example 62-Fluoro-6-[2-propyl-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]phenoxy]benzoic acid disodium salt hydrate

[0092]

[0093] A. Preparation of 2-fluoro-6-[2-propyl-3-[3-[4-bromo-2-ethyl-5-(phenylmethoxy)phenoxy]propoxy]phenoxy]-benzoic acid methylester.

[0094] 2-Fluoro-6-(3-hydroxy-2-propylphenoxy)benzoic acid methyl ester(1.84 g, 4.80 mmol) was alkylated with 2-benzyloxy-1-bromo-5-ethyl-4-(3-chloro-1-propyloxy)benzene to provide crudeproduct as an oil. Silica gel chromatography provided 2.05 g (66%) ofthe purified title intermediate as a colorless oil: NMR (CDCl₃) 7.49 (d,J=7.1 Hz, 2H), 7.20-7.45 (m, 5H), 7.14 (t, J=8.2 Hz, 1H), 6.82 (t, J=8.5Hz, 1H), 6.73 (d, J=8.3 Hz, 1H), 6.60 (d, J=8.4 Hz, 1H), 6.53 (s, 1H),6.52 (d, J=8.5 Hz, 1H), 5.13 (s, 2H), 4.20 (t, J=6.0 Hz, 2H), 4.13 (t, J=6.0 Hz, 2H), 3.92 (s, 3H), 2.58 (m, 4H), 2.30 (quintet, J =6.0 Hz, 2H),1.51 (hextet, J=7.6 Hz, 2H), 1.16 (t, J=7.9 Hz, 3H), 0.90 (t, J=7.3 Hz,3H).

[0095] B. Preparation of 2-fluoro-6-[2-propyl-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]phenoxy]-benzoic aciddisodium salt hydrate. To a solution of2-fluoro-6-[2-propyl-3-[3-[4-bromo-2-ethyl-5-(phenylmethoxy)phenoxy]propoxy]phenoxy]benzoic acid methyl ester(1.77 g, 2.72 mmol) in benzene (12 mL) was addedtetrakis(triphenylphosphine)palladium(0) (0.33 g, 0.30 mmol) and 2.0 Maqueous sodium carbonate (4 mL). To this mixture was added a solution of4-fluorophenylboronic acid (4.10 g, 8.16 mmol) in ethanol (5 mL). Theresulting mixture was refluxed for 4 hours then cooled to roomtemperature. The mixture was diluted with ethyl acetate and shaken. Theorganic layer was washed once with water and once with 1N aqueous sodiumhydroxide, dried over sodium sulfate, filtered, and concentrated invacuo to provide an oil. The product was de-benzylated, hydrolysed,salified, and purified to provide 403 mg (25%) of the desired titleproduct as a fluffy white solid: NMR (DMSO-d₆) 9.83 (bs, 1H), 7.50 (m,2H), 6.96-7.16 (m, 4H), 6.96 (s, 1H), 6.74 (t, J=8.4 Hz, 2H), 6.57 (s,1H), 6.40 (d, J=8.3 Hz, 1H), 6.35 (d, J=8.3 Hz, 1H), 4.16 (t, J=5.7 Hz,2H), 4.05 (t, J=5.5 Hz, 2H), 2.40-2.58 (m, 4H), 2.18 (quintet, J=4.1 Hz,2H), 1.41 (hextet, J=7.4 Hz, 2H), 1.07 (t, J=7.5 Hz, 3H), 0.80 (t, J=7.3Hz, 3H); MS-FAB m/e 586 (p +1, 35), 585 (p, 100), 562 (33), 313 (30). IR(CHCl₃, cm⁻¹) 3300 (b), 2967, 1616, 1455, 1398, 1115.

[0096] Analysis for C₃₃H₃₁O₆F₂Na·H₂O: Calc: C, 65.77; H, 5.52; Found: C,65.81; H, 5.41.

Example 74-Fluoro-2-[2-propyl-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]phenoxy]benzoic acid

[0097]

[0098] A. Preparation of 4-fluoro-2-(3-hydroxy-2-propyl-phenoxy) benzoicacid methyl ester.

[0099] To a solution of 2-propylresorcinol (10.0 g, 65.7 mmol) inpyridine (120 mL;) was added potassium tert-butoxide (7.00 g, 62.5 mmol)at room temperature with stirring. To this was added a mixture of methyl2-bromo-4-fluorobenzoate (7.60 g, 32.6 mmol) and copper(I) iodide (12.5g, 65.7 mmol) in pyridine (120 mL). The resulting mixture was gentlyrefluxed for 4 hours. The reaction was cooled to room temperature andstirred for 18 hours. The mixture was concentrated in vacuo and theresulting material dissolved in ethyl ether. The solution was washedonce with 5N aqueous hydrochloric acid. The aqueous layer was extractedonce with fresh ethyl ether and the combined organic layers were washedtwice with 5N aqueous ammonium hydroxide. The organic layer was washedonce with a saturated sodium chloride solution, dried over sodiumsulfate, filtered, and concentrated in vacuo. Silica gel chromatographyof the resulting residue provided 1.45 g (15%) of the desiredintermediate product as a light tan solid: mp 92-94° C.; NMR (CDCl₃)7.95 (m, 1H), 7.04 (t, J=9.5 Hz, 1H), 6.79 (t, J=9 Hz, 1H), 6.65 (d,J=9.5 Hz, 1H), 6.50 (m, 2H), 5.25 (bs, 1H, —OH), 3.88 (s, 3H), 2.60 (t,J=8.7 Hz, 2H), 1.55 (hextet, J=7.8 Hz, 2H), 0.92 (t, J=7.8 Hz, 3H);MS-FD m/e 305 (p +1, 40), 304 (p, 100); IR.

[0100] Analysis for C₁₇H₁₇O₄F: Calc: C, 67.10; H, 5.63; Found: C, 67.32;H, 5.78.

[0101] B. Preparation of 4-fluoro-2-[2-propyl-3-[3-[4-(4-fluorophenyl)-2-ethyl-5-(phenylmethoxy)phenoxy]propoxy]-phenoxy]benzoic acid methylester.

[0102] 4-Fluoro-6-(3-hydroxy-2-propylphenoxy)benzoic acid methyl ester(0.534 g, 1.75 mmol) was alkylated with 2-benzyloxy-1-(4-fluorophenyl)-5-ethyl-4-(3-chloro-1-propyloxy) benzene to providecrude product as an oil. Purification via silica gel chromatographyprovided 640 mg (55%) of the desired title intermediate as a whitecrystalline solid: mp 77-78° C.; NMR (CDCl₃) 7.95 (t, J=7.8 Hz, 1H),7.53 (m, 2H), 7.32 (m, 4H), 7.03-7.20 (m, 3H), 6.77 (m, 2H), 6.62 (s,1H), 6.55 (d, J=8 Hz, 1H), 6.50 (d, J=9 Hz, 1H), 5.05 (s, 2H), 4.25 (m,4H), 3.89 (s, 3H), 2.65 (m, 4H), 2.34 (quintet, J=6 Hz, 4H), 1.55(hextet, J=6 Hz, 2H), 1.22 (t, J=7 Hz, 3H), 0.92 (t, J=7 Hz, 3H); MS-FDm/e 666 (p); IR (CHCl₃, cm⁻¹) 2960, 1730, 1600, 1499, 1461, 1268, 1110.

[0103] Analysis for C₄₄H₄₀O₆F₂: Calc: C, 73.86; H, 6.05; Found: C,73.17; H, 6.44.

[0104] C. Preparation of 4-fluoro-2-[2-propyl-3-[3-[4-(4-fluorophenyl)-2-ethyl-5-hydroxyphenoxy]propoxy]phenoxy]-benzoic acid methyl ester.

[0105]4-Fluoro-2-[2-propyl-3-[3-[4-(4-fluorophenyl)-2-ethyl-5-(phenylmethoxy)phenoxy]propoxy]phenoxy]benzoicacid methyl ester (590 mg) was dissolved in ethyl acetate (25 mL)containing 10% palladium on carbon (118 mg) and hydrogenated at 2atmospheres for 18 hours. The mixture was filtered through Celite® andconcentrated in vacuo to provide an oil. Purification of the crudematerial via silica gel chromatography provided 400 mg (79%) of thetitle intermediate as a glass: NMR (CDCl₃) 7.97 (t, J=7.8 Hz, 1H), 7.44(m, 2H), 7.17 (m, 3H), 7.03 (s, 1H), 6.79 (m, 2H), 6.45-6.63 (m, 3H),5.38 (bs, 1H, -OH), 4.22 (m, 4H), 3.92 (s, 3H), 2.65 (m, 4H), 2.35(quintet, J=5 Hz, 2H), 1.57 (hextet, J=7 Hz, 2H), 1.24 (t, J=7.8 Hz,3H), 0.95 (t, J=7.8 Hz, 3H); MS-FD m/e 578 (p +2, 50), 577 (p +1, 90),576 (p, 100); IR (CHCl₃, cm⁻¹) 3563 (b), 2965, 1722, 1604, 1585, 1497,1461, 1267, 1251, 1152, 1110.

[0106] Analysis for C₃₄H₃₄O₆F₂: Calc: C, 70.82; H, 5.94; Found: C,71.12; H, 5.96.

[0107] D. Preparation of 4-fluoro-2-[2-propyl-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]-phenoxy]benzoic acid.

[0108] 4-Fluoro-2-[2-propyl-3-[3-[4-(4-fluorophenyl)-2-ethyl-5-hydroxyphenoxy]propoxy]phenoxy]benzoic acid methyl ester (350 mg) washydrolyzed to provide 310 mg (91%) of the desired title product as awhite solid: mp 62-64° C.; NMR (CDCl₃) 8.21 (t, J=7.8 Hz, 1H), 7.35 (m,2H), 7.10-7.30 (m, 3H), 7.97 (s, 1H), 6.84 (m, 2H), 6.63 (d, J=6.8 Hz,1H), 6.52 (s, 1H), 6.41 (d, J=9 Hz, 1H), 5.10 (bs, 1H, —OH), 4.23 (m,4H), 2.57 (m, 4H), 2.34 (quintet, J=5 Hz, 2H), 1.50 (hextet, J=6 Hz,2H), 1.17 (t, J=7.8 Hz, 3H), 0.88 (t, =7.8 Hz, 3H); MS-FD m/e 564 (p +2,30), 562 (p, 100); IR (CHCl₃, cm⁻¹) 3379 (b), 2963, 1699, 1607, 1500,1268, 1247, 1146, 1110, 839.

[0109] Analysis for C₃₃H₃₂O₆F₂: Calc: C, 70.45; H, 5.73; Found: C,70.15; H, 5.81.

Example 8 2-[2-Propyl-3-[5-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]pentoxy]phenoxy]benzoic acid

[0110]

[0111] A. Preparation of2-(5-chloropentoxy)-4-(phenyl-methoxy)acetophenone.

[0112] A mixture of 2-hydroxy-4-(phenylmethoxy)acetophenone (15.5 g,64.0 mmol), potassium carbonate (8.83 g, 64.0 mmol), anddimethylsulfoxide (15 mL) in 2-butanone (145 mL) was stirred at roomtemperature for 30 minutes. 1-Bromo-5-chloropentane (11.9 g, 64.0 mmol)was added and the resulting mixture heated at reflux for 18 hours. Thereaction mixture was cooled, diluted with water, and extracted withethyl acetate. The organic layer was washed once with a saturated sodiumchloride solution, dried over sodium sulfate, filtered, and concentratedin vacuo to provide a waxy solid. Purification via silica gelchromatography (ethyl acetate/hexane) provided 16.1 g (73%) of the titleintermediate as a white solid: mp 76-77° C.; NMR (CDCl₃) 7.85 (d, J=8.7Hz, 1H), 7.43 (m, 5H), 6.59 (d, J=8.5 Hz, 1H), 6.53 (s, 1H), 5.11 (s,2H), 4.05 (t, J=6 Hz, 2H), 3.61 (t, J=6 Hz, 2H), 2.60 (s, 3H), 1.90 (m,4H), 1.69 (m, 2H); MS-FD m/e 348 (p +2, 65), 346 (p, 100); IR (CHCl₃,cm⁻¹) 3025, 1662, 1598, 1268, 1184, 1139, 1027.

[0113] B. Preparation of2-(5-chloropentoxy)-4-(phenyl-methoxy)ethylbenzene.

[0114] To a solution of 2-(5-chloropentoxy)-4-(phenylmethoxy)-acetophenone (15.0 g, 43.2 mmol) in trifluoroacetic acid (33.3 mL) at0° C. was added triethylsilane (11.0 g, 95.1 mmol) dropwise. Theresulting mixture was stirred at 0° C. for 2.5 hours then treated withexcess saturated sodium bicarbonate solution. The mixture was extractedwith ether. The organic layer was washed once with a saturated sodiumchloride solution, dried over sodium sulfate, filtered, and concentratedin vacuo to reveal a yellow oil. Purification via silica gelchromatography (ethyl acetate/hexane) provided 10.45 g (73%) of thetitle intermediate as a faint yellow oil: NMR (CDCl₃) 7.20-7.55 (m, 5H),7.08 (d, J=9.7 Hz, 1 H), 6.53 (s, 1H), 6.51 (d, J=8.7 Hz, 1H), 5.05 (s,2H), 3.95 (t, J=6.8 Hz, 2H), 3.60 (t, J=6.8 Hz, 2H), 2.59 (q, J=7.8 Hz,2H), 1.75-1.95 (m, 4H), 1.69 (quintet, J=6 Hz), 1.18 (t, J=7.8 Hz, 3H);MS-FD m/e; IR (CHCl₃, cm⁻¹) 2937, 1613, 1587, 1505, 1289, 1258, 1172,1132, 1028.

[0115] Analysis for C₂₀H₂₅O₂Cl: Calc: C, 72.12; H, 7.57; Found: C,71.24; H, 7.64.

[0116] C. Preparation of3-bromo-6-(5-chloropentoxy)-4-(phenylmethoxy)ethylbenzene.

[0117] A mixture of 2-(5-chloropentoxy)-4-(phenylmethoxy)-ethylbenzene(10.0 g, 31.0 mmol) and N-bromosuccinimide (5.35 g, 30.1 mmol) in carbontetrachloride (100 mL) was warmed slightly for 2 hours, then stirred atroom temperature for 18 hours. The mixture was washed sequentially withwater, IN aqueous sodium thiosulfate solution, and saturated sodiumchloride solution. The organic layer was dried over sodium sulfate,filtered, and concentrated in vacuo to provide a white solid.Recrystallization from hexane provided 10.0 g (81%) of the desired titleintermediate as a white crystalline solid: mp 54-55° C.; NMR (CDCl₃)7.50 (m, 2H), 7.25-7.48 (m, 4H), 6.48 (s, 1H), 5.15 (s, 2H), 3.91 (t,J=6 Hz, 2H), 3.58 (t, J=6 Hz, 2H), 2.55 (q, J=7 Hz, 2H), 1.85 (m, 4H),1.65 (m, 2H), 1.16 (t, J=7.8 Hz, 3H); MS-FD m/e 414 (p +2, 25), 412 (p,100), 410 (p −2, 85); IR (CHCl₃, cm⁻¹) 2950, 1602, 1501, 1450, 1370,1300, 1163.

[0118] Analysis for C₂₀H₂₄O₂BrCl: Calc: C, 58.34; H, 5.87; Found: C,58.31; H, 6.04.

[0119] D. Preparation of 6-(5-chloropentoxy)-2-(4-fluorophenyl)-4-(phenylmethoxy)ethylbenzene.

[0120] 3-Bromo-6-(5-chloropentoxy)-4-(phenylmethoxy)-ethylbenzene (8.80g, 26.4 mmol) was coupled to 4-fluorophenyl-boronic acid. Purificationvia silica gel chromatography (ethyl acetate/hexane) followed byrecrystallization from hexane provided 7.04 g (77%) of the intermediatetitle product as a white solid: mp 55-56° C.; NMR (CDCl₃) 7.54 (m, 2H),7.33 (m, 5H), 7.11 (m, 3H), 6.59 (s, 1H), 5.07 (s, 2H), 3.99 (t, J=6 Hz,2H), 3.62 (t, J=6 Hz, 2H), 2.65 (q, J=8 Hz, 2H), 1.90 (m, 4H), 1.70 (m,2H), 1.14 (t, J=8 Hz, 3H); IR (CHCl₃, cm⁻¹) 2938, 1613, 1497, 1143,1027.

[0121] Analysis for C₂₆H₂₈O₂ClF: Calc: C, 73.14; H, 6.61; Found: C,72.91; H, 6.69.

[0122] E. Preparation of 2-[2-propyl-3-[5-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]pentoxy]phenoxy]-benzoic acid methyl ester.

[0123] 2-(3-Hydroxy-2-propylphenoxy)benzoic acid methyl ester (2.00 g,6.99 mmol) was alkylated with6-(5-chloropentoxy)-2-(4-fluorophenyl)-4-(phenylmethoxy)ethylbenzene toprovide crude product as an oil. Purification via silica gelchromatography (ethyl acetate/hexane) provided 3.90 g (83%) of titleintermediate as a colorless oil: NMR (CDCl₃) 7.94 (d, J=8 Hz, 1H), 7.55(m, 2H), 7.35 (m, 6H), 7.11 (m, 5H), 6.85 (d, J=9 Hz, 1H), 6.70 (d, J=9Hz, 1H), 6.60 (s, 1H), 6.48 (d, J=9 Hz, 1H), 5.07 (s, 2H), 4.08 (t, J=5Hz, 2H), 4.03 (t, J=5 Hz, 2H), 3.89 (s, 3H), 2.70 (m, 4H), 1.95 (m, 4H),1.76 (m, 2H), 1.62 (m, 2H), 1.24 (t, J=7 Hz, 3H), 0.95 (t, J=7 Hz, 3H);MS-FD m/e 677 (p +1, 65), 676 (p, 100); IR (CHCl₃, cm⁻¹) 2965, 1740,1604, 1497, 1461, 1453, 1306, 1111.

[0124] Analysis for C₄₂H₄₅O₆F: Calc: C, 76.31; H, 6.70; Found: C, 76.24;H, 6.83.

[0125] F. Preparation of 2-[2-propyl-3-[5-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]pentoxy]phenoxy]benzoic acid.

[0126] 2-[2-Propyl-3-[5-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]pentoxy]phenoxy]benzoic acid methyl ester (3.60 g, 5.32 mmol)was submitted to de-benzylation and hydrolysis. The resulting productwas isolated via vacuum filtration as a white crystalline solid: mp 65°C. (dec); NMR (CDCl₃) 8.25 (dd, J=7.9, 1.7 Hz, 1H), 7.44 (m, 3H), 7.18(m, 4H), 6.97 (s, 1H), 6.80 (d, J=8.2 Hz, 1H), 6.75 (d, J=8.5 Hz, 1H),6.65 (d, J=8.1 Hz, 1H), 6.54 (s, 1H), 5.15 (bs, 1H, —OH), 4.10 (t, J=6.1Hz, 2H), 4.05 (t, J=5.6 Hz, 2H), 2.61 (m, 4H), 1.93 (m, 4H), 1.75 (m,2H), 1.54 (hextet, J=7.4 Hz, 2H), 1.18 (t, J=7.4 Hz, 3H), 0.89 (t, J=7.3Hz, 3H); MS-FD m/e 572 (p); IR (CHCl₃, cm⁻¹) 3350 (b), 2965, 1739, 1605,1496, 1455, 1238, 1108.

[0127] Analysis for C₃₅H₃₇O₆F: Calc: C, 73.41; H, 6.51; Found: C, 73.13;H, 6.59.

Example 92-[2-Propyl-3-[4-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)-phenoxy]butoxy]phenoxy]benzoicacid sesquihydrate

[0128]

[0129] A. Preparation of2-(4-chlorobutoxy)-4-(phenyl-methoxy)acetophenone.

[0130] 2-Hydroxy-4-(phenylmethoxy)acetophenone (9.20 g, 37.9 mmol) wasalkylated with 1-bromo-4-chlorobutane. The crude material was purifiedvia silica gel chromatography (ethyl acetate/hexane) to provide 7.70 g(61%) of the desired title product as a white solid: mp 58-60° C.; NMR(CDCl₃) 7.83 (d, J=9 Hz, 1H), 7.33-7.47 (m, 5H), 6.59 (dd, J=9, 2 Hz,1H), 6.53 (d, J=2 Hz, 1H), 5.10 (s, 2H), 4.05 (t, J=5 Hz, 2H), 3.62 (t,J =5 Hz, 2H), 2.57 (s, 3H), 2.02 (m, 4H); MS-FD m/e 334 (p +1, 50), 333(p, 28), 332 (p −1, 100); IR (CHCl₃, cm⁻¹) 3013, 1663, 1599, 1267, 1184,1027.

[0131] Analysis for C₁₉H₂₁O₃Cl: Calc: C, 68.57; H, 6.36; Found: C,68.77; H, 6.60.

[0132] B. Preparation of2-(4-chlorobutoxy)-4-(phenyl-methoxy)ethylbenzene.

[0133] 2-(4-Chlorobutoxy)-4-(phenylmethoxy)-acetophenone (3.50 g, 10.5mmol) was reduced. Purification via silica gel chromatography (ethylacetate/hexane) provided 2.60 g (79%) of the desired title intermediateas a colorless oil: NMR (CDCl₃) 7.13-7.55 (m, 5H), 7.08 (d, J=8.9 Hz,1H), 6.54 (m, 2H), 5.07 (s, 2H), 3.99 (d, J=5.7 Hz, 2H), 3.65 (t, J=6.0Hz, 2H), 2.65 (q, J=7.5 Hz, 2H), 2.00 (m, 4H), 1.22 (t, J=7.5 Hz, 3H);MS-FD m/e; IR (CHCl₃, cm⁻¹) 2966, 1613, 1506, 1289, 1171, 1132, 1028.

[0134] Analysis for C₁₉H₂₃O₂Cl: Calc: C, 71.57; H, 7.27; Found: C,71.78; H, 7.40.

[0135] C. Preparation of3-bromo-6-(4-chlorobutoxy)-4-(phenylmethoxy)ethylbenzene.

[0136] 2-(4-Chlorobutoxy)-4-(phenylmethoxy)ethylbenzene (2.50 g, 7.84mmol) was brominated. Recrystallization of the crude product from hexaneprovided 2.52 g (81%) of the desired title product: mp 65-66° C.; NMR(CDCl₃) 7.50 (d, J=8 Hz, 2H), 7.34-7.48 (m, 3H), 7.32 (s, 1H), 6.49 (s,1H), 5.15 (s, 2H), 3.92 (t, J=5.6 Hz, 2H), 3.64 (t, J=5.9 Hz, 2H), 2.55(q, J=7.5 Hz, 2H), 1.97 (m, 4H), 1.15 (t, J=7.5 Hz, 3H); MS-FD m/e 398(p, 100), 396 (p −2, 70); IR (CHCl₃, cm⁻¹) 2967, 1602, 1501, 1455, 1389,1285, 1163, 1060.

[0137] Analysis for C₁₉H₂₂O₂BrCl: Calc: C, 57.38; H, 5.57; Found: C,57.27; H, 5.62.

[0138] D. Preparation of 6-(4-chlorobutoxy)-2-(4-fluorophenyl)-4-(phenylmethoxy)ethylbenzene.

[0139] 3-Bromo-6-(4-chlorobutoxy)-4-(phenylmethoxy)-ethylbenzene (2.30g, 26.4 mmol) was coupled to 4-fluorophenylboronic acid. Purificationvia silica gel chromatography (ethyl acetate/hexane) followed bytrituration with methanol provided 2.07 g (87%) of the titledintermediate product as a white solid: mp 48-49° C.; NMR (CDCl₃) 7.55(m, 2H), 7.35 (m, 5H), 7.12 (m, 3H), 6.59 (s, 1H), 5.08 (s, 2H), 4.03(t, J=5.3 Hz, 2H), 3.68 (t, J=5.3 Hz, 2H), 2.67 (q, J=7.5 Hz, 2H), 2.02(m, 4H), 1.24 (t, J=7.5 Hz, 3H); MS-FD m/e 412 (p); IR.

[0140] Analysis for C₂₅H₂₆O₂ClF: Calc: C, 72.72; H, 6.35; Found: C,72.59; H, 6.46.

[0141] E. Preparation of 2-[2-propyl-3-[4-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]butoxy]phenoxy]-benzoic acid methyl ester.

[0142] 2-(3-Hydroxy-2-propylphenoxy)benzoic acid methyl ester (1.40 g,4.84 mmol) was alkylated with6-(4-chlorobutoxy)-2-(4-fluorophenyl)-4-(phenylmethoxy)ethylbenzene toprovide crude product as an oil. Purification via silica gelchromatography (ethyl acetate/hexane) provided 2.40 g (75%) of the titleintermediate as a colorless oil: NMR (CDCl₃) 7.93 (dd, J=6.2, 1.7 Hz,1H), 7.54 (m, 2H), 7.25-7.45 (m, 6H), 7.13 (m, 5H), 6.88 (d, J=8.8 Hz,1H), 6.70 (d, J =8.8 Hz, 1H), 6.63 (s, 1H), 6.50 (d, J=8.3 Hz, 1H), 5.07(s, 2H), 4.12 (m, 4H), 3.89 (s, 3H), 2.68 (m, 4H), 2.09 (m, 4H), 1.63(hextet, J=7.4 Hz, 2H), 1.15 (t, J=7.4 Hz, 3H), 0.97 (t, J=7.4 Hz, 3H);MS-FD m/e 663 (p +1, 35), 662 (p, 100); IR (CHCl₃, cm⁻¹) 3470, 2950,1760, 1740, 1461, 1305, 1135, 1071.

[0143] Analysis for C₄₂H₄₃O₆F: Calc: C, 76.11; H, 6.54; Found: C, 76.36;H, 6.65.

[0144] F. Preparation of 2-[2-propyl-3-[4-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]butoxy]phenoxy]benzoic acid sesquihydrate.

[0145] 2-[2-Propyl-3-[4-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]butoxy]phenoxy]benzoic acid methyl ester (2.20 g, 3.32 mmol) wassubmitted to de-benzylation and hydrolysis. This procedure provided 1.00g (85%) of the title product as a white solid: mp 65-68° C.; NMR (CDCl₃)8.26 (dd, J=6.0, 1.8 Hz, 1H), 7.43 (m, 3H), 7.12-7.29 (m, 4H), 6.99 (s,1H), 6.81 (d, J=8 Hz, 1H), 6.75 (d, J=8.2 Hz, 1H), 6.65 (d, J=8 Hz, 1H),6.53 (s, 1H), 5.08 (bs, 1H, —OH), 4.12 (m, 4H), 2.63 (m, 4H), 2.08 (m,4H), 1.55 (hextet, J=7.4 Hz, 2H), 1.20 (t, J=7.4 Hz, 3H), 0.90 (t, J=7.4Hz, 3H); MS-FD m/e 559 (p +1, 57), 558 (p, 100); IR (CHCl₃, cm⁻¹) 3350(b), 2950, 1739, 1625, 1496, 1455, 1237, 1108.

[0146] Analysis for C₃₄H₃₅O₆F·1.5 H₂O: Calc: C, 69.73; H, 6.43; Found:C, 69.74; H, 6.54.

Example 102-[2-(2-Methylpropyl)-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]phenoxy]benzoic acid

[0147]

[0148] A. Preparation of 2-(2-methylpropyl)-1,3-dimethoxy-benzene.

[0149] To a solution of 1,3-dimethoxybenzene (38.0 g, 272 mmol) intetrahydrofuran (380 mL) at 0° C. was added a 1.6 M solution ofbutyllithium in hexane (188 mL, 299 mmol). The resulting mixture wasstirred at 0° C. for 2 hours. 1-Iodo-2-methylpropane (50.0 g, 272 mmol)was added and the reaction mixture warmed to room temperature, thenrefluxed for 36 hours. The mixture was cooled to room temperature,diluted with saturated ammonium chloride solution, and extracted twicewith ethyl acetate. The organic layer was dried over sodium sulfate,filtered, and concentrated in vacuo. Purification via silicachromatography (ethyl acetate/hexane) provided 13.8 g (26%) of titleintermediate product as a colorless oil: NMR (CDCl₃) 7.22 (t, J=9 Hz,1H), 6.33 (d, J=10 Hz, 2H), 3.89 (s, 6H), 2.66 (d, J=9 Hz, 2H), 2.03(heptet, J=8 Hz, 1H), 1.00 (d, J=8 Hz, 6H); IR (CHCl₃, cm⁻¹) 2959, 1593,1474, 1261, 1133, 1075.

[0150] B. Preparation of 2-(2-methylpropyl)-1,3-dihydroxybenzene.

[0151] 2-(2-Methylpropyl)-1,3-dimethoxybenzene (18.0 g, 92.8 mmol) wasmelted with pyridinium hydrochloride (90 g) and stirred at 180° C. for 8hours. The mixture was cooled to room temperature, diluted with water,and extracted twice with ethyl acetate. The organic phase was washedwith dilute aqueous hydrochloric acid, dried over sodium sulfate,filtered and concentrated in vacuo. Purification via silica gelchromatography (ether/hexane) provided 15.0 g (98%) of titleintermediate as a light yellow oil: NMR (CDCl₃) 6.97 (t, J=9 Hz, 1H),6.43 (d, J=10 Hz, 2H), 5.68 (s, 2H, —OH), 2.59 (d, J=9 Hz, 2H), 2.03(heptet,

[0152] J=8 Hz, 1H), 1.00 (d, J=8 Hz, 6H); MS-FD m/e 166 (p); IR (CHCl₃,cm⁻¹) 3603, 3349 (b), 2959, 1601, 1466, 1298, 1104, 987.

[0153] Analysis for C₁₀H₁₄O₂: Calc: C, 72.26; H, 8.49; Found: C, 72.37;H, 8.75.

[0154] C. Preparation of 2-[3-hydroxy-2-(2-methylpropyl)-phenoxy]benzoicacid methyl ester.

[0155] 2-(2-Methylpropyl)-1,3-dihydroxybenzene (14.5 g, 87.3 mmol) wassubmitted to Ullmann coupling conditions with methyl 2-iodobenzoate.Purification of the crude product via silica gel chromatography(ether/hexane) provided 3.11 g (12%) of the desired title intermediateas a light yellow oil: NMR (CDCl₃) 7.91 (d, J=8 Hz, 1H), 7.23 (t, J=8Hz, 1H), 7.16 (t, J=8 Hz, 1H), 6.99 (t, J=8 Hz, 1H), 6.86 (d, J=9 Hz,1H), 6.63 (d, J=9 Hz, 1H), 6.39 (d, J=9 Hz, 1H), 5.42 (bs, 1H, —OH),3.84 (s, 3H), 2.58 (d, J =9 Hz, 2H), 2.08 (heptet, J=8 Hz, 1H), 0.99 (d,J=8 Hz, 6H); MS-FD m/e 300 (p); IR (CHCl₃, cm⁻¹) 3625, 3360 (b), 2950,1718, 1602, 1453, 1306, 1235, 1107, 910.

[0156] Analysis for C₁₈H₂₀O₄: Calc: C, 71.98; H, 6.71; Found: C, 72.19;H, 6.86.

[0157] D. Preparation of 2-[2-(2-methylpropyl)-3-[3-[2-ethyl-5-(phenylmethoxy)-4-(4-fluorophenyl)phenoxy]propoxy]-phenoxy]benzoicacid methyl ester.

[0158] 2-[3-Hydroxy-2-(2-methylpropyl)phenoxy]benzoic acid methyl ester(750 mg, 2.51 mol) was alkylated with 2-benzyloxy-l-(4-fluorophenyl)-5-ethyl-4-(3-chloro-1-propyloxy) benzene to providecrude product as an oil. Purification via silica gel chromatography(ether/hexane) provided 620 mg (35%) of title intermediate product as anoff-white solid: mp 82-84° C.; NMR (CDCl₃) 7.99 (d, J=8 Hz, 1H), 7.62(t, J=7 Hz, 2H), 7.38 (m, 6H), 7.18 (m, 5H), 6.90 (d, J=9 H, 1H), 6.78(d, J=9 Hz, 1H), 6.71 (s, 1H), 6.53 (d, J=9 Hz, 1H), 5.09 (s, 2H), 4.27(m, 4H), 3.91 (s, 3H), 2.70 (m, 4H), 2.39 (quintet, J=8 Hz, 2H), 2.10(heptet, J=8 Hz, 1H), 1.30 (t, J=9 Hz, 3H), 1.00 (d, J=8 Hz, 6H); MS-FDm/e 663 (p +1, 42), 662 (p, 100); IR (KBr, cm⁻¹) 3425 (b), 2959, 2864,1733, 1604, 1580, 1500, 1447, 1246, 1080, 837.

[0159] Analysis for C₄₂H₄₃O₆F: Calc: C, 76.11; H, 6.54; Found: C, 76.20;H, 6.83.

[0160] E. Preparation of 2-[2-(2-methylpropyl)-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]phenoxy]-benzoic acid.

[0161]2-[2-(2-Methylpropyl)-3-[3-[2-ethyl-5-(phenylmethoxy)-4-(4-fluorophenyl)phenoxy]propoxy]phenoxy]benzoicacid methyl ester (600 mg, 0.906 mmol) was submitted to de-benzylationconditions. Hydrolysis of the resulting ester provided 250 mg (57%) oftitle product as an off-white solid: mp 48-49° C.; NMR (CDCl₃) 8.25 (d,J=9 Hz, 1H), 7.44 (m, 3H), 7.20 (m, 4H), 7.05 (s, 1H), 6.85 (d, J=9 Hz,1H), 6.76 (d, J=9 Hz, 1H), 6.64 (d, J=9 Hz, 1H), 6.59 (s, 1H), 5.32 (bs,1H, —OH), 4.28 (m, 4H), 2.63 (q, J=8 Hz, 2H), 2.52 (d, J=8 Hz, 2H), 2.38(quintet, J=8 Hz, 2H), 1.96 (heptet, J=8 Hz, 1H), 1.23 (t, J=9 Hz, 3H),0.98 (d, J=8 Hz, 6H); MS-FD m/e 559 (p +1, 39), 558 (p, 100); IR (KBr,cm⁻¹) 3350 (b), 2958, 1699, 1604, 1457, 1222, 1112, 1062, 838, 756.

[0162] Analysis for C₃₄H₃₅O₆F: Calc: C, 73.10; H, 6.31; Found: C, 73.32;H, 6.50.

Example 11 2-[2-Butyl-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]phenoxy]benzoic acid hydrate

[0163]

[0164] A. Preparation of 2-butyl-1,3-dimethoxybenzene.

[0165] 1,3-Dimethoxybenzene (15.0 g, 109 mmol) was alkylated with1-iodobutane as described above for the preparation of Example 10(A)except that the final reaction mixture was not refluxed. Purificationvia silica gel chromatography (ethyl acetate/hexane) provided 15.0 g(71%) of the title intermediate product as a yellow oil: NMR (CDCl₃)7.18 (t, J=8.2 Hz, 1H), 6.59 (d, J=9.7 Hz, 2H), 3.84 (s, 6H), 2.70 (t,J=8.7 Hz, 2H), 1.50 (hextet, J=6 Hz, 2H), 1.44 (quintet, J=6 Hz, 2H),0.98 (t, J=8.2 Hz, 3H); MS-FD m/e 194 (p).

[0166] B. Preparation of 2-(3-hydroxy-2-butylphenoxy)-benzoic acidmethyl ester.

[0167] 2-Butyl-1,3-dimethoxybenzene (14.98 g, 77.6 mmol) wasde-methylated as described above for the preparation of Example 10(B) toprovide 19 g crude product as a brown oil. A solution of 15 g of thismaterial and potassium tert-butoxide (9.70 g, 86.5 mmol) in pyridine(150 mL) was added to a second solution of methyl 2-iodobenzoate (11.9g, 180 mmol) and copper(I) iodide (17.3 g, 91.0 mmol) in pyridine (150mL). The resulting mixture was refluxed for 36 hours. The mixture wascooled to room temperature, diluted with water, and extracted threetimes with diethyl ether The combined ether fractions were filteredthrough a mat of Celite®, washed once with 5N aqueous hydrochloric acid,once with 2N aqueous sodium hydroxide, and filtered again through a matof Celite®. The resulting solution was dried over magnesium sulfate,filtered, and evaporated in vacuo. Silica gel chromatography (ethylacetate/hexane) provided 3.02 g (11%) of the title intermediate productas an orange oil: NMR (CDCl₃) 7.91 (d, J=8 Hz, 1H), 7.41 (t, J=8 Hz,1H), 7.14 (t, J=8 Hz, 1H), 6.97 (t, J=9 Hz, 1H), 6.83 (d, J=8 Hz, 1H),6.59 (d, J=8 Hz, 1H), 6.39 (d, J=8 Hz, 1H), 5.04 (bs, 1H, —OH), 3.83 (s,3H), 2.66 (t, J=9 Hz, 2H), 1.54 (quintet, J=5 Hz, 2H), 1.35 (hextet, J=5Hz, 2H), 0.91 (t, J=8 Hz, 3H); MS-EI m/e 300 (p, 34), 225 (100), 213(42), 197 (53), 107 (38); IR (mull, cm⁻¹) 3410, 2926, 1709, 1600, 1463,1234, 1107, 1090, 992.

[0168] Analysis for C₁₈H₂₀O₄: Calc: C, 71.98; H, 6.71; Found: C, 70.82;H, 6.67.

[0169] C. Preparation of 2-[2-butyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]propoxy]phenoxy]-benzoic acid methyl ester.

[0170] 2-(3-Hydroxy-2-butylphenoxy)benzoic acid methyl ester (700 mg,1.76 mmol) was alkylated with 2-benzyloxy-1-(4-fluorophenyl)-5-ethyl-4-(3-chloro-1-propyloxy)benzene as described above for thepreparation of Example 5(A) to provide crude product as an oil.Purification via silica gel chromatography (ethyl acetate/hexane)provided 700 mg (60%) of the title intermediate product as a yellow oil:NMR (CDCl₃) 7.91 (d, J=9 Hz, 1H), 7.58 (m, 2H), 7.38 (m, 6H), 7.18 (m,5H), 6.88 (d, J=10 Hz, 1H), 6.76 (d, J=9 Hz, 1H), 6.68 (s, 1H), 6.47 (d,J=9 Hz, 1H), 5.09 (s, 2H), 4.25 (m, 4H), 3.91 (s, 3H), 2.72 (m, 4H),2.40 (quintet, J=5 Hz, 2H), 1.60 (hextet, J=5 Hz, 2H), 1.38 (m, 2H),1.24 (t, J=8 Hz, 3H), 0.99 (t, J=8 Hz, 3H); IR (CHCl₃, cm⁻¹) 3024, 1717,1602, 1465, 1453, 1306, 1234, 1086, 1014.

[0171] Analysis for C₄₂H₄₃O₆F: Calc: C, 76.11; H, 6.54; Found: C, 75.82;H, 6.50.

[0172] D. Preparation of 2-[2-butyl-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]phenoxy]benzoic acid hydrate.

[0173] 2-[2-Butyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]propoxy]phenoxy]benzoic acid methyl ester (690 mg, 1.04 mmol)was submitted to de-benzylation conditions as described above for thepreparation of Example 7(C). Hydrolysis of the resulting ester provided114 mg (30%) of the title product as an off-white solid: mp 62-64° C.;NMR (DMSO-d₆) 12.75 (bs, 1H, —COOH), 9.60 (bs, 1H, —OH), 7.69 (d, J=7.3Hz, 1H), 7.50 (m, 2H), 7.35 (t, J=7.4 Hz, 1H), 7.00-7.18 (m, 4H), 6.96(s, 1H), 6.69 (m, 2H), 6.56 (s, 1H), 6.31 (d, J=8.2 Hz, 1H), 4.17 (t,J=5.1 Hz, 2H), 4.09 (t, J=5.4 Hz, 2H), 2.58 (t, J=7.3 Hz, 2H), 2.48 (m,2H), 2.21 (quintet, J=5.0 Hz, 2H), 1.37 (hextet, J=6.8 Hz, 2H), 1.21 (m,2H), 1.06 (t, J=7.4 Hz, 3H), 0.74 (t, J=7.1 Hz, 3H); MS-FD m/e 559 (p+1, 55), 558 (p, 100); IR (KBr, cm⁻¹) 3350 (b), 2963, 2933, 1738, 1605,1497, 1461, 1455, 1236, 1118.

[0174] Analysis for C₃₄H₃₅O₆F·H₂O: Calc: C, 70.81; H, 6.47; Found: C,71.19; H, 6.52.

Example 122-[2-(Phenylmethyl)-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]phenoxy]benzoic acid

[0175]

[0176] A. Preparation of 2-(phenylmethyl)-1,3-dimethoxybenzene.

[0177] 1,3-Dimethoxybenzene (75.0 g, 391 mmol) was alkylated with benzylbromide as described above for the preparation of Example 10(A) exceptthat the final reaction mixture was not refluxed. Purification viasilica gel chromatography (ether/hexane) provided 18.8 g (8%) ofintermediate product as a white solid: 53-55° C.; NMR (CDCl₃) 7.15-7.37(m, 6H), 6.62 (d, J=10 Hz, 2H), 4.12 (s, 2H), 3.87 (s, 6H); MS-FD m/e229 (p +1, 17), 228 (p, 100); IR (KBr, cm⁻¹) 2925, 2839, 1594, 1476,1435, 1259, 1197, 1106, 700.

[0178] Analysis for C₁₅H₁₆O₂: Calc: C, 78.92; H, 7.06; Found: C, 79.21;H, 7.33.

[0179] B. Preparation of 2-(phenylmethyl)-1,3-dihydroxybenzene.

[0180] 2-(Phenylmethyl)-1,3-dimethoxybenzene (15.0 g, 65.8 mmol) wasde-methylated as described above for the preparation of Example 10(B).Purification via silica gel chromatography (ethyl acetate/hexane)provided 7.76 g (60%) of title intermediate product as an off-whitecrystalline material: mp 81-83° C.; NMR (CDCl₃) 7.18-7.23 (m, 5H), 7.01(t, J=9 Hz, 1H), 6.43 (d, J=10 Hz, 2H), 5.38 (bs, 2H, —OH), 4.18 (s,2H); MS-FD m/e 201 (p +1, 23), 200 (p, 100); IR (KBr, cm⁻¹) 3505 (b),1618, 1464, 1360, 1292, 1183, 1012, 739.

[0181] Analysis for C₁₃H₁₂O₂: Calc: C, 77.98; H, 6.04; Found: C, 77.69;H, 5.99.

[0182] C. Preparation of 2-[3-hydroxy-2-(phenylmethyl)-phenoxy]benzoicacid methyl ester.

[0183] 2-(Phenylmethyl)-1,3-dihydroxybenzene (14.5 g, 87.3 mmol) wassubmitted to Ullmann coupling conditions with methyl 2-iodobenzoate asdescribed above for the preparation of Example 2(A). Purification of thecrude product via silica gel chromatography (ethyl acetate/hexane)provided 900 mg (7%) of title intermediate product as a whitecrystalline material: mp 79-81° C.; NMR (CDCl₃) 7.93 (d, J=9 Hz, 1H),7.35 (m, 3H), 7.27 (m, 2H), 7.13 (m, 2H), 7.04 (d, J=9 Hz, 1H), 6.83 (d,J=9 Hz, 1H), 6.63 (d, J=9 Hz, 1H), 6.41 (d, J=9 Hz, 1H), 5.43 (bs, 1H,—OH), 4.14 (s, 2H), 3.79 (s, 3H); MS-FD m/e 335 (p +1, 23), 334 (p,100); IR (KBr, cm⁻¹) 3327 (b), 1687, 1598, 1453, 1315, 1233, 1008, 754.

[0184] Analysis for C₂₁H₁₈O₄: Calc: C, 75.43; H, 5.43; Found: C, 75.21;H, 5.57.

[0185] D. Preparation of 2-[2-(phenylmethyl)-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]propoxy]-phenoxy]benzoicacid methyl ester.

[0186] 2-[3-Hydroxy-2-(phenylmethyl)phenoxy]benzoic acid methyl ester(840 mg, 2.51 mmol) was alkylated with 2-benzyloxy-1-(4-fluorophenyl)-5-ethyl-4-(3-chloro-1-propyloxy) benzene asdescribed above for the preparation of Example 5(A). Purification viasilica gel chromatography (ethyl acetate/hexane) provided 680 mg (40%)of desired title intermediate product as a glass: NMR (CDCl₃) 8.01 (d,J=8 Hz, 1H), 7.65 (m, 2H), 7.40 (m, 8H), 7.15-7.30 (m, 8H), 6.88 (d,J=10 Hz, 1H), 6.80 (d, J=10 Hz, 1H), 6.63 (s, 1H), 6.48 (d, J=9 Hz, 1H),5.09 (s, 2H), 4.34 (t, 7 Hz, 2H), 4.22 (s, 2H), 4.20 (t, J=7 Hz, 2H),3.84 (s, 3H), 2.77 (q, J=8 Hz, 2H), 2.40 (quintet, J=8 Hz, 2H), 1.38 (t,J=9 Hz, 3H); MS-FD m/e 698 (p +1, 48), 697 (p, 100); IR (CHCl₃, cm⁻¹)3015, 2975, 1717, 1604, 1496, 1453, 1306, 1081.

[0187] Analysis for C₄₅H₄₁O₆F: Calc: C, 77.57; H, 5.93; Found: C, 77.80;H, 6.08.

[0188] E. Preparation of 2-[2-(phenylmethyl)-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]phenoxy]-benzoic acid.

[0189]2-[2-(Phenylmethyl)-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]propoxy]phenoxy]benzoicacid methyl ester (660 mg, 0.947 mmol) was submitted to de-benzylationconditions and hydrolysis. Purification via silica gel chromatography(ethyl acetate/hexane) provided 450 mg (80%) the desired title productas a glass: NMR (CDCl₃) 8.16 (dd, J=7.8, 1.8 Hz, 1H), 7.43 (m, 2H), 7.35(m, 1H), 7.05-7.32 (m, 9H), 7.02 (s, 1H), 6.86 (d, 8.4 Hz, 1H), 6.66 (d,J=8.4 Hz, 1H), 6.61 (d, J=8.2 Hz, 1H), 6.46 (s, 1H), 4.28 (t, J=4.6 Hz,2H), 4.10 (t, J=4.1 Hz, 2H), 4.08 (s, 2H), 2.64 (q, J=7.5 Hz, 2H), 2.33(quintet, J=5.1 Hz, 2H), 1.22 (t, J=7.5 Hz, 3H); MS-FD m/e 593 (p, 100),592 (p - 1, 89); IR (CHCl₃, cm⁻¹) 3375 (b), 3020, 2970, 1738, 1605,1496, 1455, 1068.

[0190] Analysis for C₃₇H₃₃O₆F: Calc: C, 74.98; H, 5.61; Found: C, 75.21;H, 5.72.

Example 13 2-[2-Propyl-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]benzoyl]benzoic acid

[0191]

[0192] A. Preparation of 2-[3-(allyloxy)benzoyl]benzoic acid.

[0193] To a solution of 3-(allyloxy)bromobenzene (15.0 g, 70.5 mmol) intetrahydrofuran (750 mL) at -70° C. was added 1.6M n-butyllithium (44.1mL, 70.5 mmol). After stirring for 1 hour, a solution of phthalicanhydride (11.4 g, 77.0 mmol) in tetrahydrofuran (100 mL, previouslycooled to −70° C.) was added over 1 hour. The mixture was allowed towarm to room temperature and stirred for 3 hours. The mixture wasdiluted with saturated ammonium chloride solution and extracted withdiethyl ether. The organic layer was washed three times with 1N sodiumhydroxide solution and the combined aqueous layers were back-extractedwith a fresh portion of diethyl ether. The aqueous layer was adjusted topH˜3 with aqueous hydrochloric acid and extracted three times with freshdiethyl ether. The combined organic layers were washed once with water,once with saturated sodium chloride solution, dried over sodium sulfate,filtered, and concentrated in vacuo to reveal an off-white solid.Recrystallization from ether/hexane provided 10.3 g (52%) of the titleintermediate as a white crystalline material: mp 109° C.; NMR (CDCl₃)8.20 (d, J=8 Hz, 1H), 7.65 (t, J=8 Hz, 1H), 7.60 (t, J=8 Hz, 1H),7.30-7.45 (m, 3H), 7.28 (d, J=8 Hz, 1H), 7.20 (d, J=8 Hz, 1H), 6.02 (m,1H), 5.35 (d, J=16 Hz, 1H), 5.30 (d, J=11 Hz, 1H), 4.55 (d, J=6 Hz, 2H);MS-FD m/e 283 (p +1, 27), 282 (p, 100).

[0194] Analysis for C₁₇H₁₄O₄: Calc: C, 72.33; H, 5.00; Found: C, 72.07;H, 5.22.

[0195] B. Preparation of 2-[3-(allyloxy)benzoyl]benzoic acid methylester.

[0196] A solution of 2-[3-(allyloxy)benzoyl]benzoic acid (9.00 g, 31.9mmol) in methanol (100 mL) was saturated with hydrogen chloride gas. Theresulting solution was stirred at room temperature for 18 hours. Thereaction mixture was concentrated in vacuo and diluted with diethylether. The resulting solution was washed sequentially with a saturatedsodium bicarbonate solution, water, and a saturated sodium chloridesolution. The organic layer was dried over sodium sulfate, filtered, andconcentrated in vacuo. The resulting pale yellow oil solidified uponstanding to provide 9.45 g (100%) of the desired title product as awhite solid: mp 50-52° C.; NMR (CDCl₃) 8.05 (d, J=7.8 Hz, 1H), 7.65 (t,J=8 Hz, 1H), 7.56 (t, J=8 Hz, 1H), 7.40 (m, 2H), 7.32 (t, J=8 Hz, 1H),7.22 (d, J=8 Hz, 1H), 7.14 (d, J=8 Hz, 1H), 6.08 (m, 1H), 5.40 (d, J=16Hz, 1H), 5.30 (d, J=11 Hz, 1H), 4.78 (d, J=4 Hz, 2H), 3.62 (s, 3H);MS-FD m/e 297 (p +1, 40), 296 (p, 100); IR.

[0197] Analysis for C₁₈H₁₆O₄: Calc: C, 72.46; H, 5.44; Found: C, 72.75;H, 5.58.

[0198] C. Preparation of 2-[3-hydroxy-2-[3-(1-propenyl)]-benzoyl]benzoicacid methyl ester and 2-[3-hydroxy-4-[3-(l-propenyl)]benzoyl]benzoicacid methyl ester.

[0199] 2-[3-(Allyloxy)benzoyl]benzoic acid methyl ester (6.70 g, 20.2mmol) was heated neat at 175° C. for 30 hours. The product mixture wascooled to room temperature and purified via silica gel chromatography(95:5 methylene chloride/ethyl acetate) to provide 3.62 g (54%) of2-[3-hydroxy -2-[3-(1-propenyl)]-benzoyl]benzoic acid methyl ester and1.44 g (21%) of 2-[3-hydroxy-4-[3-(1-propenyl)]enzoyl]benzoic acidmethyl ester as white solids.

[0200] 2-[3-Hydroxy-2-[3-(1-propenyl)]benzoyl]benzoic acid methyl ester,mp 107-109° C.; NMR (CDCl₃) 7.91 (dd, J=7.8, 2.2 Hz, 1H), 7.43-7.63 (m,3H), 7.08 (m, 1H), 7.02 (d, J=8 Hz, 1H), 6.80 (dd, J=8, 2 Hz, 1H), 6.15(m, 1H), 5.42 (bs, 1H, —OH), 5.23 (d, J=16 Hz, 1H), 5.16 (d, J=11 Hz,1H), 3.81 (d, J=6 Hz, 2H), 3.68 (s, 3H); MS-FD m/e 297 (p +1, 40), 296(p, 100), 278 (45); IR.

[0201] Analysis for C₁₈H₁₆O₄: Calc: C, 72.96; H, 5.44; Found: C, 73.26;H, 5.54.

[0202]2-[3-Hydroxy-4-[3-(1-propenyl)]benzoyl]benzoic acid methyl ester,mp 139-140° C.; NMR (CDCl₃) 8.08 (dd, J=7.9, 3.1 Hz, 1H), 7.63 (t, J=8Hz, 1H), 7.55 (t, J=8 Hz, 1H), 7.40 (d, J=8 Hz, 1H), 7.35 (s, 1H), 7.16(s, 2H), 6.00 (m, 1H), 5.62 (bs, 1H, —OH), 5.15 (m, 2H), 3.65 (s, 3H),3.47 (d, J=5 Hz, 2H); MS-FD m/e 297 (p +1, 20), 296 (p, 100); IR.

[0203] Analysis for C₁₈H₁₆O₄: Calc: C, 72.96; H, 5.44; Found: C, 73.11;H, 5.50.

[0204] D. Preparation of 2-[2-[3-(l-propenyl)]-3-[3-[2-ethyl-5-(phenylmethoxy)-4-(4-fluorophenyl)phenoxy]propoxy]-benzoyl]benzoicacid methyl ester.

[0205] 2-[3-Hydroxy-2-[3-(1-propenyl)]benzoyl]benzoic acid methyl ester(520 mg, 1.75 mmol) was alkylated with 2-benzyloxy-1-(4-fluorophenyl)-5-ethyl-4-(3-chloro-1-propyloxy) benzene asdescribed above for the preparation of Example 5(A). Recrystallizationof the crude product from ether/hexane provided 750 mg (65%) of thedesired title intermediate as a white solid: mp 90-91° C.; NMR (CDCl₃)7.91 (m, 1H), 7.53 (m, 4H), 7.45 (m, 1H), 7.32 (m, 5H), 7.02-7.22 (m,5H), 6.85 (d, J=8 Hz, 1H), 6.61 (s, 1H), 6.10 (m, 1H), 5.04 (d, J=16 Hz,1H), 5.03 (s, 2H), 4.99 (d, J=11 Hz, 1H), 4.23 (m, 4H), 3.77 (d, J=7 Hz,2H), 3.66 (s, 3H), 1.64 (q, J=6 Hz, 2H), 2.37 (quintet, J=6 Hz, 2H),1.19 (t, J=8 Hz, 3H); MS-FD m/e 659 (p +1, 44), 658 (p, 100).

[0206] Analysis for C₄₂H₃₉O₆F: Calc: C, 76.58; H, 5.97; Found: C, 76.79;H, 6.09.

[0207] E. Preparation of 2-[2-propyl-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]benzoyl]benzoic acid.

[0208]2-[2-[3-(1-Propenyl)]-3-[3-[2-ethyl-5-(phenylmethoxy)-4-(4-fluorophenyl)phenoxy]propoxy]benzoyl]benzoicacid methyl ester (318 mg, 0.483 mmol) was submitted to hydrogenationconditions as described above for the preparation of Example 7(C).Hydrolysis of the resulting ester and purification via silica gelchromatography (ethyl acetate/hexane) provided 150 mg (56%) of the titleproduct as a glass: NMR (DMSO-d₆) 10.15 (bs, 1H, —OH), 7.84 (m, 1H),7.49 (m, 2H), 7.41 (m, 2H), 6.98-7.23 (m, 5H), 6.96 (s, 1H), 6.62 (d,J=7.2 Hz, 1H), 6.59 (s, 1H), 4.18 (t, J=5.3 Hz, 2H), 4.06 (t, J=5.8 Hz,2H), 2.85 (m, 2H), 2.49 (m, 2H), 2.20 (quintet, J=5.2 Hz, 2H), 1.57(hextet, J=5 Hz, 2H), 1.08 (t, J=7.4 Hz, 3H), 0.90 (t, J=7.2 Hz, 3H);IR, MS.

[0209] Analysis for C₃₄H₃₃O₆F: Calc: C, 73.36; H, 5.98; Found: C, 69.71:H, 5.90.

Example 14 2-[[2-Propyl-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]phenyl]methyl]benzoic acid

[0210]

[0211] A. Preparation of 2-[(3-hydroxy-2-propylphenyl)-methyl]benzoicacid methyl ester.

[0212] A mixture of 2-[3-hydroxy-2-[3-(1-propenyl)]benzoyl]-benzoic acidmethyl ester (3.00 g, 10.1 mmol), concentrated sulfuric acid (1 mL), and5% palladium on carbon (1.5 g) in methanol (95 mL) was hydrogenated at 4atmospheres for 18 hours. The mixture was concentrated in vacuo to avolume of approximately 30 mL, filtered, and saturated with hydrogenchloride gas. The resulting mixture was stirred for 18 hours, thenconcentrated in vacuo. The residue was dissolved in diethyl ether andwashed with a saturated sodium bicarbonate solution. The aqueous layerwas back-extracted with a fresh portion of diethyl ether. The combinedorganic layers were washed with a saturated sodium chloride solution,dried, filtered, and concentrated in vacuo to provide 2.60 g (90%) ofthe title intermediate as an orange oil: NMR (CDCl₃) 7.97 (d, J=7 Hz,1H), 7.38 (t, J=7 Hz, 1H), 7.28 (t, J=7 Hz, 1H), 7.02 (m, 2H), 6.70 (d,J=7 Hz, 1H), 6.54 (d, J=7 Hz, 1H), 5.20 (bs, 1H, —OH), 4.45 (s, 2H),3.89 (s, 3H), 2.58 (t, J=7 Hz, 2H), 1.52 (hextet, J=7 Hz, 2H), 0.92 (t,J=7 Hz, 3H); MS-FD m/e 285 (p +1, 23), 284 (100); IR.

[0213] B. Preparation of 2-[[2-propyl-3-[3-[2-ethyl-5-(phenylmethoxy)-4-(4-fluorophenyl)phenoxy]propoxy]phenyl]-methyl]benzoic acid methylester.

[0214] 2-[(3-Hydroxy-2-propylphenyl)methyl]benzoic acid methyl ester(2.00 g, 4.68 mmol) was alkylated with 2-benzyloxy-1-(4-fluorophenyl)-5-ethyl-4-(3-chloro-1-propyloxy) benzene asdescribed above for the preparation of Example 5(A). Recrystallizationof the crude product from hexane provided 1.72 g (38%) of the titleintermediate as a white solid: mp 83-84° C.; NMR (CDCl₃) 7.94 (d, J=8Hz, 1H), 7.53 (m, 2H), 7.25-7.40 (m, 7H), 7.05-7.15 (m, 4H), 7.00 (d,J=7 Hz, 1H), 7.81 (d, J=7 Hz, 1H), 6.62 (s, 1H), 6.58 (d, J=7 Hz, 1H),5.02 (s, 2H), 4.42 (s, 2H), 4.21 (m, 4H), 3.88 (s, 3H), 2.54-2.68 (m,4H), 2.32 (quintet, J=6 Hz, 2H), 1.50 (hextet, J=6 Hz, 2H), 1.21 (t, J=8Hz, 3H), 0.96 (t, J=8 Hz, 3H); MS-FD m/e 648 (p +1, 40), 647 (p, 100);IR (CHCl₃, cm⁻¹) 2964, 1718, 1603, 1497, 1459, 1143.

[0215] Analysis for C₄₂H₄₃O₅F: Calc: C, 77.99; H, 6.70; Found: C, 79.47;H, 6.76.

[0216] C. Preparation of 2-[[2-propyl-3-[3-[2-ethyl-5-hydroxy-4-(4-fluorophenyl)phenoxy]propoxy]phenyl]methyl]-benzoic acid.

[0217] 2-[[2-Propyl-3-[3-[2-ethyl-5-(phenylmethoxy)-4-(4-fluorophenyl)phenoxy]propoxy]phenyl]methyl]benzoic acid methyl ester (1.50 mg, 2.32mmol) was submitted to de-benzylation conditions as described above forthe preparation of Example 7(C). Hydrolysis of the resulting esterfollowed by recrystallization of the crude product from ether/hexaneprovided 860 mg (68%) of the desired title product as a white solid: mp150-151° C. ; NMR (CDCl₃) 8.11 (dd, J=7.3, 0.8 Hz, 1H), 7.45 (m, 2H),7.30 (t, J=7 Hz, 1H), 6.95-7.25 (m, 5H), 6.81 (d, J=8.0 Hz, 1H), 6.58(d, J=7.4 Hz, 1H), 6.52 (s, 1H), 4.50 (s, 2H), 4.21 (m, 4H), 2.62 (m,4H), 2.35 (quintet, J=6.0 Hz, 2H), 1.46 (hextet, J=7.6 Hz, 2H), 1.18 (t,J=7.5 Hz, 3H), 0.93 (t, J=7.4 Hz, 3H); MS-FD m/e 543 (p +1, 40), 542 (p,100); IR (CHCl₃, cm⁻¹) 3400 (b), 2966, 1696, 1603, 1496, 1459, 1238,1146, 1111.

[0218] Analysis for C₃₄H₃₅O₅F: Calc: C, 75.26; H, 6.50; Found: C, 75.26;H, 6.62.

Example 152-[2-Propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]thiophenoxy]benzoicacid

[0219]

[0220] A. Preparation of 2-bromophenyldisulfide.

[0221] To a mixture of 2-bromothiophenol (20.0 g, 106 mmol) and 2Nsodium hydroxide solution (100 mL) in diethyl ether (400 mL) was addedsolid iodine (13.4 g, 53.0 mmol) in portions. The mixture was stirred atroom temperature for 1 hour at which time the ether layer was separated.The aqueous layer was extracted with a fresh portion of ether and thecombined ether layers were washed once with water, once with a saturatedsodium chloride solution, dried over sodium sulfate, filtered, andconcentrated in vacuo to provide 17.2 g (43%) of intermediate product asa white solid: mp 95-97° C.; NMR (CDCl₃) 7.52 (m, 4H), 7.25 (t, J=9.7Hz, 2H), 7.06 (t, J=9.7 Hz, 2H); MS-FD m/e 380 (p +4, 20), 379 (p +3,30), 378 (p +2, 85), 376 (p, 100), 374 (p −2, 75); IR.

[0222] Analysis for C₁₂H₈Br₂S₂: Calc: C, 38.32; H, 2.14; Found: C,38.61; H, 2.13.

[0223] B. Preparation of 2-[3-(allyloxy)thiophenoxy]-bromobenzene.

[0224] To a solution of 3-(allyloxy)bromobenzene (8.20 g, 38.7 mmol) intetrahydrofuran (600 mL) at -74° C. was added 1.6 M n-butyllithium (24.2mL, 38.7 mmol). After stirring for 30 minutes this solution wascannulated into a solution of 2-bromophenyl-disulfide (16.0 g, 42.5mmol) in tetrahydrofuran (160 mL) at −74° C. The resulting mixture wasallowed to warm to room temperature then diluted with saturatedammonium-chloride solution and filtered. The aqueous layer was extractedwith three times with diethyl ether and the combined organic layers werewashed once with water, once with a saturated sodium chloride solution,dried over sodium sulfate, filtered, and concentrated in vacuo toprovide a yellow oil. Purification via silica gel chromatographyprovided 9.40 g (76%) of the title intermediate as a light yellow oil:NMR (CDCl₃) 7.58 (d, J=7 Hz, 1H), 7.27 (t, J=7 Hz, 1H), 7.17 (t, J=7 Hz,1H), 6.85-7.15 (m, 5H), 6.04 (m, 1H), 5.41 (d, J=14 Hz, 1H), 5.30 (d,J=10 Hz, 1H), 4.52 (d, J=4 Hz, 2H); MS-FD m/e 322 (p, 100), 320 (p, 75);IR (KBr, cm⁻¹) 3223 (b), 1688, 1345, 1161, 1013, 678.

[0225] Analysis for C₁₅H₁₃OBrS: Calc: C, 56.09; H, 4.08; Found: C,56.31; H, 4.22.

[0226] C. Preparation of 2-[3-(allyloxy)thiophenoxy]-benzoic acid methylester.

[0227] To a solution of 2-[3-(allyloxy)thiophenoxy]-bromobenzene (9.00g, 28.0 mmol) in tetrahydrofuran (175 mL) at −78° C. was added 1.6 Mn-butyllithium (19.2 mL, 30.8 mmol) dropwise. After stirring for 15minutes, the solution was saturated with carbon dioxide gas resulting ina thick gel. Tetrahydrofuran (50 mL) was added and the resulting mixtureallowed to warm to room temperature. The mixture was diluted withsaturated ammonium chloride solution. The aqueous layer was extractedonce with diethyl ether and the combined organic layers wereconcentrated in vacuo. The residue was dissolved in a fresh portion ofether and extracted with 1N aqueous sodium hydroxide. The aqueous layerwas washed with a fresh portion of ether and acidified with aqueoushydrochloric acid. The resulting aqueous layer was extracted with afresh portion of ether. The organic layer was washed with a saturatedsodium chloride solution, dried over sodium sulfate, filtered, andconcentrated in vacuo. The crude acid was dissolved in methanol (125 mL)and the resulting solution saturated with hydrogen chloride gas. Afterstirring for 18 hours, the reaction mixture was concentrated in vacuo,the residue dissolved in ether, and the resulting solution washed withsaturated sodium bicarbonate solution. The aqueous layer wasback-extracted with a fresh portion of ether and the combined organiclayers were washed once with water, once with a saturated sodiumchloride solution, dried over sodium sulfate, filtered, and concentratedin vacuo. Purification via silica gel chromatography (ethylacetate/hexane) provided 4.80 g (68%) of the desired title intermediateas a faint yellow oil: NMR (CDCl₃) 7.99 (dd, J=7.8, 1.4 Hz, 1H), 7.33(t, J=7 Hz, 1H), 7.25 (t, J=7 Hz, 1H), 7.15 (m, 3H), 7.00 (dd, J=8.7,2.8 Hz, 1H), 6.88 (d, J=8 Hz, 1H), 6.04 (m, 1H), 5.42 (d, J=14 Hz, 1H),5.30 (d, J=11 Hz, 1H), 4.53 (d, J=3.9 Hz, 2H), 3.97 (s, 3H); MS-FD m/e301 (p +1, 25), 300 (p, 100); IR (CHCl₃, (cm⁻¹) 3025, 1712, 1590, 1463,1437, 1254, 1060.

[0228] Analysis for C₁₇H₁₆O₃S: Calc: C, 67.98; H, 5.37; Found: C, 67.86;H, 5.29.

[0229] D. Preparation of2-[3-hydroxy-2-[3-(l-propenyl)]-thiophenoxy]benzoic acid methyl esterand 2-[3-hydroxy-4-[3-(1-propenyl)]thiophenoxy]benzoic acid methylester.

[0230] 2-[3-(Allyloxy)thiophenoxy]benzoic acid methyl ester (5.40 g,15.0 mmol) was heated neat at 175° C. for 29 hours. The product mixturewas cooled to room temperature and purified via silica gelchromatography (methylene chloride) to provide 2.22 g (41%) of2-[3-hydroxy-2-[3-(1-propenyl)]thio-phenoxy]benzoic acid methyl esterand 1.46 g (27%) of 2-[3-hydroxy-4-[3-(1-propenyl)]thiophenoxy]benzoicacid methyl ester as white solids.

[0231] 2-[3-Hydroxy-2-[3-(1-propenyl)]thiophenoxy]benzoic acid methylester, mp 72-74° C.; NMR (DMSO-d₆) 9.79 (s, 1H, —OH), 7.89 (d, J=8 Hz,1H), 7.33 (t, J=7 Hz, 1H), 7.09-7.23 (m, 2H), 6.94 (m, 2H), 6.62 (dd,J=7, 1 Hz, 1H), 5.78 (m, 1H), 4.70-4.83 (m, 2H), 3.86 (s, 3H), 3.37 (d,J=5 Hz, 2H); MS-FD m/e 301 (p +1, 21), 300 (p, 100); IR (CHCl₃, cm⁻¹)3595, 3350 (b), 3029, 3010, 2954, 1711, 1420, 1436, 1273, 1146, 1060.

[0232] Analysis for C₁₇H₁₆O₃S: Calc: C, 67.98; H, 5.37; Found: C, 68.28;H, 5.41.

[0233] 2-[3-Hydroxy-4-[3-(1-propenyl)]thiophenoxy]benzoic acid methylester, mp 96-97° C.; NMR (DMSO-d₆) 9.78 (s, 1H, —OH), 7.89 (d, J=8 Hz,1H), 7.40 (t, J=7 Hz, 1H), 7.12-7.25 (m, 2H), 6.93 (s, 1H), 6.91 (d, J=8Hz, 1H), 6.81 (d, J=8 Hz, 1H), 5.87 (m, 1H), 5.00-5.12 (m, 2H), 3.85 (s,3H), 3.30 (d, J=4 Hz, 2H); MS-FD m/e 301 (p +1, 45), 300 (p, 100); IR(CHCl₃, cm⁻¹) 3595, 3300(b), 3029, 3010, 2954, 1711, 1436, 1310, 1255,942. Calc: C, 67.98; H, 5.37; Found: C, 68.04; H, 5.47.

[0234] E. Preparation of 2-[2-[3-(1-propenyl)]-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]propoxy]-thiophenoxy]benzoicacid methyl ester.

[0235] 2-[3-Hydroxy-2-[3-(1-propenyl)]thiophenoxy]benzoic acid methylester (2.00 g, 6.66 mmol) was alkylated with 2-benzyloxy-1-(4-fluorophenyl)-5-ethyl-4-(3-chloro-1-propyloxy) benzene asdescribed above for the preparation of Example 5(A). Purification viasilica gel chromatography (hexane/diethyl ether) provided 2.90 g (66%)of desired intermediate product as a white solid: mp 76-77° C.; NMR(CDCl₃) 8.03 (dd, J=7.6, 1.2 Hz, 1H), 7.54 (m, 2H), 7.17-7.40 (m, 8H),6.98-7.18 (m, 5H), 6.71 (d, J=7.9 Hz, 1H), 6.62 (s, 1H), 5.87 (m, 1H),5.03 (s, 2H), 4.83-4.95 (m, 2H), 4.26 (t, J=7 Hz, 2H), 4.21 (t, J=7 Hz,2H), 3.98 (s, 3H), 3.62 (d, J=6.3 Hz, 2H), 2.64 (q, J=7.5 Hz, 2H), 2.33(quintet, J=5.8 Hz, 2H), 1.22 (t, J=7.5 Hz, 3H); MS-FD m/e 664 (p +2,40), 663 (p +1, 70), 662 (p, 100); IR (CHCl₃, cm⁻¹) 3011, 2970, 2940,2890, 1712, 1497, 1452, 1298, 1255, 1145, 1060.

[0236] Analysis for C₄₁H₃₉O₅FS: Calc: C, 74.30; H, 5.93; Found: C,74.46; H, 6.13.

[0237] F. Preparation of 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]thiophenoxy]benzoic acid methyl ester.

[0238] 2-[2-[3-(1-Propenyl)]-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-(phenylmethoxy)phenoxy]propoxy]-thiophenoxy]benzoic acid methyl ester(2.70 g, 4.07 mmol) was hydrogenated as described above for thepreparation of Example 7(C) to provide an oil (˜2 g). A solution of thismaterial (1.39 g) in methylene chloride (25 mL) at −78° C. was treatedwith 1M boron tribromide (3.61 mL, 3.61 mmol) and allowed to stir for 1hour. The reaction mixture was diluted with water and extracted withmethylene chloride. The organic layer was washed with water, dried oversodium sulfate, filtered, and concentrated in vacuo to provide a yellowoil. Purification via silica gel chromatography provided 770 mg (47%) ofthe title intermediate as a white solid: mp 105-106° C.; NMR (CDCl₃)8.02 (dd, J=7.6, 1.2 Hz, 1H), 7.43 (m, 2H), 7.07-7.30 (m, 8H), 6.98 (m,2H), 6.71 (d, J=7.9 Hz, 1H), 6.57 (s, 1H), 5.10 (bs, 1H, —OH), 4.24 (m,2H), 3.98 (s, 3H), 2.83 (t, J=7 Hz, 2H), 2.65 (q, J=7.5 Hz, 2H), 2.36(quintet, J=5 Hz, 2H), 1.52 (hextet, J=6 Hz, 2H), 1.21 (t, J=7.4 Hz,3H), 0.90 (t, J=7.5 Hz, 3H); MS-FD m/e 575 (p +1, 20), 574 (p, 100); IR.

[0239] G. Preparation of 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]thiophenoxy]benzoic acid.

[0240]2-[2-Propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]thiophenoxy]benzoicacid methyl ester (700 mg, 1.22 mmol) was hydrolyzed to provide 689 mg(100%) of the desired title product as a white solid: mp 153-155° C.;NMR (CDCl₃) 8.13 (dd, J=8.2, 0.9 Hz, 1H), 7.42 (m, 2H), 7.10-7.33 (6H),6.99 (m, 2H), 6.72 (d, J=7.9 Hz, 1H), 6.55 (s, 1H), 4.90 (bs, 1H, —OH),4.24 (m, 4H), 2.82 (t, J=6 Hz, 2H), 2.63 (q, J=7.5 Hz, 2H), 2.34(quintet, J=6 Hz, 2H), 1.51 (hextet, J=7.5 Hz, 2H), 1.18 (t, J=7.5 Hz,3H), 0.90 (t, J=7.5 Hz, 3H); MS-FD m/e 561 (p +1, 20), 560 (p, 100); IR(CHCl₃, cm⁻¹) 2967, 1700, 1603, 1497, 1451, 1147, 1043.

[0241] Analysis for C₃₃H₃₃O₅FS: Calc: C, 70.69; H, 5.93; Found: C,70.43; H, 5.97.

Example 162-[2-Propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]phenylsulfinyl]benzoicacid

[0242]

[0243] To a solution of 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]thiophenoxy]benzoic acid (450 mg, 0.803 mmol)in methylene chloride (10 mL) at −78° C. was added a solution of 85%m-chloroperoxybenzoic acid (138 mg) in methylene chloride (2 mL). After40 minutes the mixture was concentrated in vacuo. Purification of theresidue via silica gel chromatography (95% chloroform/4.5% methanol/0.5%acetic acid) provided 380 mg (80%) of the title product as an off-whitesolid: mp >100° C. (dec) ; NMR (CDCl₃) 8.53 (d, J=8 Hz, 1H) , 8.14 (d,J=8 Hz, 1H), 7.93 (t, J=8 Hz, 1H), 7.63 (t, J=8 Hz, 1H), 7.43 (m, 2H),7.13 (m, 2H), 6.94-7.06 (m, 2H), 6.88 (d, J=8 Hz, 1H), 6.50 (d, J=8 Hz,1H), 7.46 (s, 1H), 6.38 (bs, 1H, —OH), 4.15 (m, 4H), 3.32 (m, 1H), 3.08(m, 1H), 2.57 (q, J=7.5 Hz, 2H), 2.29 (quintet, J=6 Hz, 2H), 1.75 (m,2H), 1.17 (t, J=7.5 Hz, 3H), 1.05 (t, J=7.3 Hz, 3H); MS (highresolution) calc 577.202642 (MH+), found 577.203800; IR (CHCl₃, cm⁻¹)2969, 1708, 1497, 1455, 1266, 1146, 1018.

[0244] Analysis for C₃₃H₃₃O₆FS: Calc: C, 68.73; H, 5.77; Found: C,67.54; H, 5.69.

Example 172-[2-Propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]phenylsulfonyl]benzoicacid hydrate

[0245]

[0246] To a solution of 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]phenylsulfinyl]-benzoic acid (150 mg, 0.260mmol) in methylene chloride (3.0 mL) at 0° C. was added a solution of85% m-chloroperoxybenzoic acid (53 mg) in methylene chloride (1 mL).After 1 hour the mixture was warmed to 4° C. and stirred for 18 hours.The mixture was concentrated in vacuo; purification of the residue viasilica gel chromatography (90% chloroform/9.5% methanol/0.5% aceticacid) provided 90 mg (58%) of the title product as a white solid: mp80-90° C.; NMR (DMSO-d₆) 7.88 (m, 2H), 7.55-7.78 (m, 3H), 7.50 (m, 2H),7.33 (m, 2H), 7.04 (m, 2H), 6.95 (s, 1H), 6.51 (s, 1H), 4.19 (t, J=4.8Hz, 2H), 4.05 (t, J=5.8 Hz, 2H), 2.69 (m, 2H), 2.44 (q, J=5.8 Hz, 2H),2.19 (m, 2H), 0.90-1.10 (m, 5H), 0.71 (t, J=4.5 Hz, 3H); MS-FD m/e 595(p +2, 30), 594 (p +1, 40), 593 (p, 100); IR (CHCl₃, cm⁻¹) 2966, 1730,1603, 1497, 1299, 1146.

[0247] Analysis for C₃₃H₃₃O₇FS·H₂O: Calc: C, 64.90; H, 5.78; Found: C,64.89; H, 5.67.

Example 18 8-Propyl-7-[3-[4-acetyl-3-methoxy-2-propylphenoxy]propoxy]-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid

[0248]

[0249] The title compound was prepared in a manner analogous to that ofExample 1 of U.S. Pat. No. 4,889,871.

[0250] Assay for Reversal of Adriamycin Resistance

[0251] HL60/ADR is a continuous cell line, which was selected forAdriamycin™ resistance by culturing HL60, a human acute myeloblasticleukemia cell line, in increasing concentrations of Adriamycin™ until ahighly resistant variant was attained. (McGrath, T., et al., Biochem.Pharmacol., 38: 3611, (1989); Marquardt, D. and Center, M. S., CancerRes., 52: 3157, (1992); and Marquardt, D., et al., Cancer Res., 50:1426, (1990))

[0252] HL60/ADR cells were grown in RPMI 1640 (Gibco) containing 10%fetal bovine serum (FBS) and 250 μg/ml gentamicin™ (Sigma). Cells wereharvested; washed twice with assay medium (same as culture media);counted; and diluted to 2×10⁵ cells/ml in assay medium. Fifty μl ofcells were aliquoted into wells of a 96 well tissue culture plate. Onecolumn of each 96 well plate served as a negative control and receivedassay medium containing no cells.

[0253] Test compounds and references compounds were dissolved indimethyl sulfoxide (DMSO) at a concentration of 5 mM. Samples werediluted to 20 μM in assay medium and 25 μl of each test compound wasadded to 6 wells. Assay standards were run in quadruplicate. 25 μl of0.4% DMSO was added to four wells as a solvent control. Assay media wasadded to all wells to achieve a final volume of 100 μl per well.

[0254] The plates were incubated at 37° Centigrade for 72 hours in ahumidified incubator with a 5% CO₂ atmosphere. A CellTiter96 AqueousAssay Kit (Promega) was used to measure cell viability and vitality viaoxidation of a tetrazolium salt. The assay was performed in accordancewith the vendors instructions, which required 2 ml of MTS reagent beingmixed with 0.1 ml of PMS reagent and 20 μl of this solution was added toeach well. The plates were incubated for 3 hours at 37° C. Absorbancewas determined at 490 nm using a microtitre plate reader.

[0255] The ability of a test compound to reverse the resistance ofHL60/ADR cells to Adriamycin was determined by comparison of theabsorbance of the wells containing a test compound in addition toAdriamycin with the absorbance of wells containing Adriamycin without atest compound. Controls were used to eliminate background and to ensurethe results were not artifactual. The results of the assay are expressedas percent inhibition of cell growth. Adriamycin alone at the testedconcentration does not usually inhibit the growth of HL60/ADR cells.TABLE 1 Example % Inhibition 1 54% 2 63% 3 55% 4 38% 5 23% 6 35% 7 72% 857% 9 61% 10  49% 11  47% 12  62% 13  64% 14  58% 15  73% 16  35% 17 25% 18  67%

[0256] The compounds or formulations employed in the present inventionmay be administered by the oral and rectal routes, topically,parenterally, e.g., by injection and by continuous or discontinuousintra-arterial infusion, in the form of, for example, tablets, lozenges,sublingual tablets, sachets, cachets, elixirs, gels, suspensions,aerosols, ointments, for example, containing from 1 to 10% by weight ofthe active compound in a suitable base, soft and hard gelatin capsules,suppositories, injectable solutions and suspensions in physiologicallyacceptable media, and sterile packaged powders adsorbed onto a supportmaterial for making injectable solutions. Advantageously for thispurpose, compositions may be provided in dosage unit form, preferablyeach dosage unit containing from about 5 to about 500 mg (from about 5to 50 mg in the case of parenteral or inhalation administration, andfrom about 25 to 500 mg in the case of oral or rectal administration) ofa compound of Formula I. Dosages from about 0.5 to about 300 mg/kg perday, preferably 0.5 to 20 mg/kg, of active ingredient may beadministered although it will, of course, readily be understood that theamount of the compound or compounds of Formula I actually to beadministered will be determined by a physician, in the light of all therelevant circumstances including the condition to be treated, the choiceof compound to be administered and the choice of route of administrationand therefore the above preferred dosage range is not intended to limitthe scope of the present invention in any way.

[0257] The formulations employed in the present invention normally willconsist of at least one compound of Formula I mixed with a carrier, ordiluted by a carrier, or enclosed or encapsulated by-an ingestiblecarrier in the form of a capsule, sachet, cachet, paper or othercontainer or by a disposable container such as an ampoule. A carrier ordiluent may be a solid, semi-solid or liquid material which serves as avehicle, excipient or medium for the active therapeutic substance. Someexamples of the diluents or carrier which may be employed in thepharmaceutical compositions of the present invention are lactose,dextrose, sucrose, sorbitol, mannitol, propylene glycol, liquidparaffin, white soft paraffin, kaolin, fumed silicon dioxide,microcrystalline cellulose, calcium silicate, silica,polyvinylpyrrolidone, cetostearyl alcohol, starch, modified starches,gum acacia, calcium phosphate, cocoa butter, ethoxylated esters, oil oftheobroma, arachis oil, alginates, tragacanth, gelatin, syrup, methylcellulose, polyoxyethylene sorbitan monolaurate, ethyl lactate, methyland propyl hydroxybenzoate, sorbitan trioleate, sorbitan sesquioleateand oleyl alcohol and propellants such as trichloromonofluoromethane,dichlorodifluoromethane and dichlorotetrafluoroethane. In the case oftablets, a lubricant may be incorporated to prevent sticking and bindingof the powdered ingredients in the dies and on the punch of thetableting machine. For such purpose there may be employed for instancealuminum, magnesium or calcium stearates, talc or mineral oil.

[0258] Preferred pharmaceutical forms of the present invention arecapsules, tablets, suppositories, and injectable solutions. Especiallypreferred are formulations for oral ingestion.

Example 19 Preparation of Membrane Vesicles

[0259] HL60/ADR cells (human leukemia cells grown in adriamycin andoverexpressing MRP), revertant HL60 cells, and parental HL60 cells,respectively, were harvested by centrifugation after cultivation in cellculture and plasma membrane vesicles were prepared according to standardmethods. Briefly, the cells were lysed by incubation in hypotonic buffer(0.5 mM sodium phosphate (pH 7.0), 0.1 mM EDTA supplemented withprotease inhibitors) for 1.5 hours, followed by homogenization in ahomogenizer. After centrifugation of the homogenate at 12,000 ×g(4° C.,10 minutes), the supernatant was centrifuged at 100,000 ×g at 4° C. for45 minutes. The resulting pellet was homogenized in incubation buffer(250 mM sucrose-10 mM tris-HCl, pH 7.4) and layered over 38% sucrose in5 mM 4(2-hydroxyethyl) -1-piperazineethanesulfonic acid/KOH, pH 7.4.After centrifugation at 280,000 ×g at 4° C. for 2 hours, the interphasewas collected, washed by centrifugation in incubation buffer (100,000×g) and passed 20 times through a 27-gauge needle. The resultingmembrane vesicles had enriched plasma membranes with moderatecontaminations from endoplasmic reticulum and Golgi's apparatus.

Example 20 ATP-Dependent Transport

[0260] The ATP-dependent transport of [³H]LTC₄ and S—(2,4-dinitrophenyl)[³H]glutathione into membrane vesicles was measured by rapid filtration.Membrane vesicles (50 μg of protein) were incubated in the presence of 4mM ATP, 10 mM MgCl_(2,) 10 mM of creatine phosphate, and the labeledsubstrate in an incubation buffer containing 250 mM sucrose and 10 mMTris-HCl, pH 7.4. The final volume was 110 μl. Aliquots of 20 μl weretaken at predetermined times and diluted in 1 ml of ice-cold incubationbuffer. The dilute samples were filtered immediately through anitrocellulose membrane (0.2 μm pore size), presoaked in incubationbuffer using a rapid filtration device and rinsed twice with 5 ml ofincubation buffer. The filters were immersed in liquid and measured in aliquid scintillation counter. ATP was replaced by an equal concentrationof 5′-AMP in a control experiment. All rates of the ATP-dependenttransport were obtained by subtracting the values obtained in thepresence of 5′-AMP from those measured in the presence of ATP.

[0261] The active ATP-dependent transport of [³H]LTC₄, which took placeinto the fraction of the “inside out” -oriented vesicles, wasinvestigated during a 3-minute period (FIG. 1). Using vesicles from theparental as well as the revertant cells, which both exhibited a low MRPexpression, the rate of the ATP-dependent transport of [³H]LTC₄ at thestandard concentration of 50 nM was below 1 pmol ×mg protein⁻¹ ×min⁻¹.The HL60/ADR cells, which overexpressed MRP, showed rapid ATP dependenttransport of LTC₄ (50 nM) at an initial transport rate of 25±2 pmol ×mgprotein⁻¹ ×min⁻¹ (FIG. 1). The amount of [³H]LTC₄ taken up by thevesicles was markedly decreased by the growing osmolarity of theextravesicular medium. Thus [³H]LTC₄ was transported into theintravesicular space.

[0262] The right-hand portion of FIG. 1 shows that the ATP-dependenttransport of S—(2,4-dinitrophenyl-[³H]glutathione at a concentration of5 μM using HL60/ADR membranes was 50±4 pmol ×mg protein⁻¹ ×min⁻¹. Thetransport rate of the revertant and parental HL60 cells was 10 timesless. FIG. 4 further reveals that the receptor antagonist MK 571 is apotent inhibitor of the ATP-dependent LTC₄ transport.

Example 21 Photoaffinity Labeling

[0263] Vesicle suspensions (200 μg protein) were incubated with 74 kBq[³H]LTC₄ (150 nM) at 37° C. for 10 minutes, shock-frozen in liquidnitrogen and irradiated at 300 nm for 5 minutes. For the purpose ofcompetition studies, the vesicle preparations were preincubated with 50μM of MK 571 at 4° C. for 30 minutes. The photoaffinity labeling with370 kBq of 8-azido-[∝-³²P]ATP (10 μM) was carried out in a similar wayas described above, with incubation at 4° C. for 2 minutes andirradiation at 350 nm for 10 minutes.

[0264] LTC₄-binding membrane proteins were detected by directphotoaffinity labeling using [³H]LTC₄ as a photolabile ligand. A markeddifference in the [³H]LTC₄ labeling pattern of the membranes from thedifferent cell lines was observed at a molecular weight of 190 kDa (FIG.2). In the HL60/ADR membranes, a predominant 190 kDa protein was labeled(FIG. 2, Panel A), whereas in the membranes from the revertant andparental cells only a slight labeling was detected in this range (FIG.2, Panels B and C). The [³H]LTC₄ labeling of the 190 kDa protein wascompetitively suppressed by the transport-inhibitor MK 571 (FIG. 2,Panel A). Another significantly labeled protein of 110 kDa was observedin the membranes from all three cell lines.

[0265] The photoaffinity labeling using 8-azido-[³²P]ATP also showed astrongly labeled band of a 190 kDa protein in the membranes of HL60/ADRcells but not in the revertant or parental cell membranes (FIG. 3).

[0266] A series of experiments utilizing [³H]etoposide and [³H]etoposideglucuronide were performed in substantial accordance with the method ofExample 20. The results of this series of experiments unequivocablydemonstrates MRP-mediated, ATP-dependent transport of conjugatedcytostatic drugs in membrane vesicles of MRP-overexpressingdrug-resistant human tumor cells by direct transport measurements.Reference to the Figures illustrates: the ATP-dependent transport of theetoposide glucuronide is mediated by MRP; the unconjugated etoposide isnot a substrate for MRP; and MRP-mediated ATP-dependent transport of theetoposide glucuronide is competitively inhibited by MK 571 at a 5 μMconcentration.

1. A method of treating a patient having a multidrug-resistant neoplasm,said method comprising the step of inhibiting membrane transportmediated by the multidrug resistance protein MRP by administering aneffective amount of a compound of Formula I

wherein R₁ is

Y is hydrogen or halo; R₂ is hydrogen, —OH, or —OCH₃; R₃ is C₁-C₆ alkyl;R₄ is hydrogen, —OH, or —OCH₃; n is 3, 4, or 5; A is

R₅ is hydrogen, C₁-C₆ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, benzyl, orphenyl; R₆ is hydrogen or halo; R₇ is —COOH or 5-tetrazolyl; T is abond, —CH₂—, —O—, —C(═O)—, or —S(O)_(q)—; and q is 0, 1,or 2; providedwhen one of R₂ and R₄ is —OH or —OCH₃, the other of R₂ and R₄ must behydrogen, or a pharmaceutically acceptable base addition salt or solvatethereof.
 2. The method of claim 1 wherein the compound is administeredat a concentration of about 0.5 to about 50 mg/kg body weight of thepatient.